In Vivo Evaluation of a Novel Albumin-Binding Prodrug of Doxorubicin in an Orthotopic Mouse Model of Prostate Cancer (Lncap)

Home , Doxorubicin

In vivo evaluation of a novel albumin-binding prodrug of doxorubicin in an orthotopic mouse model of prostate cancer (LNCaP)

B Elsadek1, R Graeser2, N Esser2, C Scha¨fer-Obodozie2, C Tsurumi3, K Abu Ajaj1, A Warnecke1, C Unger1, T Saleem4 and F Kratz1 1Division of Macromolecular Prodrugs, Tumor Biology Center, Freiburg, Germany; 2ProQinase GmbH, In vivo Testing, Freiburg, Germany; 3Clinic for Radiotherapy, University Hospital Freiburg, Freiburg, Germany and 4Faculty of Medicine, Department of Biochemistry, Assiut University, Assiut, Egypt

PSA, which is overexpressed in prostate carcinoma, represents a molecular target for selectively releasing an anticancer agent from a prodrug formulation. In this study, we report on the in vivo antitumor efficacy of an efficacious albumin-binding prodrug of doxorubicin (PSA9) that incorporates p-aminobenzyloxycarbonyl (PABC) as a self-immolative spacer in addition to the heptapeptide, Arg-Ser-Ser-Tyr-Tyr-Ser-Leu, which serves as a substrate for PSA. The prodrug is cleaved very efficiently by PSA releasing H-Ser-Leu-PABC-doxorubicin and subsequently doxorubicin in PSA-positive cell lysates and prostate tumor homogenates as the final cleavage product. PSA9 at 3 Â 6mgkgÀ1 doxorubicin equivalents (intravenous) was compared with conventional doxorubicin at equitoxic doses (at 3 Â 3mgkgÀ1; intravenous) in an orthotopic mouse model of prostate cancer using LNCaP lentiviral luciferase-neomycin cells transduced with luciferase. Whereas doxorubicin did not show any efficacy against the primary tumor or metastases, the prodrug reduced the primary tumor by 30–50% and circulating PSA levels, and in addition, showed a pronounced reduction in lung and bone metastases by B77% and B96%, respectively, and a positive trend regarding the activity against liver and lymph-node metastases compared with control and doxorubicin-treated animals. The incorporation of PABC as a self-immolative spacer together with a PSA substrate demonstrates superior antitumor effects over doxorubicin attributed to an efficient cleavage by PSA releasing doxorubicin as the final active agent in prostate tumor homogenates. Using this approach for developing effective prodrugs against prostate cancer, is worthy of further preclinical optimization. Prostate Cancer and Prostatic Diseases (2011) 14, 14–21; doi:10.1038/pcan.2010.43; published online 2 November 2010

Keywords: human serum albumin; PSA; orthotopic animal model

Introduction achieved with taxotere.3 Recent data obtained from two large phase III clinical trials, TAX327 and SWOG 9916, Prostate cancer is one of the leading causes of cancer- indicated that taxotere supplemented with prednisone related death in men and remains incurable in the can achieve a survival benefit in some patients with metastatic setting, especially after the formation of hormone-resistant metastatic prostate cancer, whereas osteoblastic bone metastases that remain the most lethal treatment with mitoxantrone supplemented with corti- aspect of prostate cancer.1,2 Despite the initial response to costeroids alleviated symptoms but did not improve androgen deprivation, the disease gradually progresses overall patient survival.4–6 Thus, the benefit of therapy to a hormone-refractory state as a result of cumulative with antineoplastic agents is limited because of systemic genetic alterations in tumor cells or the microenviron- toxicity and lack of tumor selectivity, and there is an ment. At present, there is no effective therapy for men urgent medical need to design drug delivery systems, with hormone-resistant metastatic prostate cancer, and which transport the anticancer agent to the primary and among the approved anticancer agents, taxotere, mitox- metastatic tumors. antrone and estramustine phosphate, the best results are In the past, we along with others have developed low- or high-molecular-weight prodrugs that are cleaved by PSA, a serine protease that is overexpressed in metastatic Correspondence: Dr F Kratz, Division of Macromolecular Prodrugs, prostate carcinoma.7–9 These prodrugs were developed Tumor Biology Center, Breisacher Strae 117, D-79106 Freiburg, Germany. E-mail: [email protected] with the aim of reducing toxicity and improving both the Received 19 August 2010; revised 20 September 2010; accepted 20 efficacy and the selectivity of chemotherapeutic agents September 2010; published online 2 November 2010 for treating hormone-refractory prostate cancer. Prodrug of doxorubicin B Elsadek et al

Over the last decade, we have intensively investigated peptide substrate with p-aminobenzyloxycarbonyl (PABC) 15 human serum albumin (HSA) as a drug carrier which is as a self-immolative spacer. having an increasing role in the clinical setting for PSA9 exhibited good water solubility (B4mgmlÀ1 in delivering anticancer drugs to the tumor.10 Uptake of isotonic 5% glucose solution), was bound rapidly to the albumin in tumors is mediated by the EPR effect, that is, cysteine-34 position of circulating HSA, and its albumin- the enhanced permeability and retention of macromole- bound form (HSA9) was cleaved efficiently by PSA cules in relation to passive tumor targeting.11 Our tumor- releasing H-Ser-Leu-PABC-DOXO.14 This doxorubicin targeting strategy that exploits albumin as a drug carrier dipeptide was further degraded in LNCaP prostate is based on two features: (1) in situ binding of a thiol- tumor tissue homogenates and in PSA-positive LNCaP binding prodrug to the cysteine-34 position of circulating luciferase-neomycin cell lysates to release doxorubicin as albumin after intravenous administration with sub- a final cleavage product within few hours by adventi- sequent accumulation of the drug albumin conjugate tious proteases.14 After cleavage and deacylation, the in the tumor due to passive targeting and (2) release of PABC spacer decomposes in 1,6-benzylelimination and the albumin-bound drug at the tumor site due to the spontaneously releases the free drug.15 incorporation of a cleavable bond between the drug Moreover, the dipeptide H-Ser-Leu-PABC-DOXO was and the carrier. Anthracyclines, such as doxorubicin, significantly more active against a PSA-expressing have frequently been used by us for the development of prostate cancer cell line (LNCaP lentiviral luciferase- such albumin-binding prodrugs.12 The first and most neomycin (LLN)) than either H-Ser-Arg-DOXO, the advanced prototype of these prodrugs is the (6-maleimi- cleavage product of PSA5, or H-Ser-Leu-DOXO that docaproyl)hydrazone derivative of doxorubicin (abbre- both do not contain the PABC spacer.14 viated DOXO-EMCH, now INNO-206), an acid-sensitive As a consequence and as a main goal of this study, we prodrug of doxorubicin that is under phase II clinical set out to investigate the antitumor efficacy of PSA9 in development13 (also see http://www.cytrx.com). comparison with conventional doxorubicin in a mouse Inspired by the translational research with DOXO- model of human prostate cancer using luciferase- EMCH, we developed a PSA-cleavable albumin-binding transduced LNCaP cells orthotopically implanted in the prodrug of doxorubicin, that is, EMC-Arg-Ser-Ser-Tyr- prostate of severe combined immunodeficiency (SCID) Tyr-Ser-Arg-DOXO (abbreviated PSA5, Figure 1a).9 PSA5 mice. As the LNCaP LLN cell line used in this study was was shown to rapidly bind to the circulating albumin genetically altered to express luciferase, tumor growth and was superior over doxorubicin in an orthotopic PSA- could be followed in vivo, and bone, lung, liver and positive model (LNCaP) with respect to antitumor inguinal lymph nodes as the most susceptible organs for efficacy on the primary tumor but showed some but the formation of metastases in progressive prostate not statistically significant activity only against lung cancer could be analysed using an in vitro luciferase metastases.9 The major drawback of this prodrug was assay. that although it was cleaved efficiently by PSA, it liberated the less active doxorubicin dipeptide H-Ser- Arg-DOXO as the major cleavage product, which only showed marginal cleavage to H-Arg-DOXO or doxo- Materials and methods rubicin in tumor homogenates of PSA-positive LNCAP tumors. All animal experiments were performed in accordance Thus, to exploit the full potential of our prodrug with the German Animal License Regulations approach, we developed an optimized albumin-binding (Tierschutzgesetz) identical to the United Kingdom prodrug of doxorubicin (abbreviated PSA9, Figure 1b), Coordinating Committee on Cancer Research guidelines which releases doxorubicin effectively as the final for the welfare of animals in experimental neoplasia.9,16 cleavage product. PSA9 combines a PSA-cleavable Male SCID (C.B-17/IcrHanHsd-Prkdc-scid) mice were

O OH O OH O O OH N H Arg-Ser-Ser-Tyr-Tyr–Ser-Arg N O OH O O O CH3 O HO Maleimide PSA cleavable peptide Doxorubicin CH3

O OH O O OH O H N N OH Arg-Ser-Ser-Tyr-Tyr–Ser-Leu H O O N O OH O O CH3 O O HO Maleimide PSA cleavable peptide PABC CH3 Doxorubicin Figure 1 Structures of the PSA-cleavable prodrugs (a) PSA5 and (b) PSA9. PABC, p-aminobenzyloxycarbonyl.

Prostate Cancer and Prostatic Diseases Prodrug of doxorubicin B Elsadek et al

16 obtained from Harlan Winkelmann GmbH (Borchen, cells, suspended in 20 ml phosphate-buffered saline, were Germany). To test novel antitumor agents in a setting injected into the anterior part of the prostate using a 29-G that reflects the human disease better than subcutaneous needle syringe. The exteriorized organs were reinserted models, we used our previously designed orthotopic into the abdomen, the abdominal wall was closed model for prostate cancer using PSA-positive luciferase- using 5-0 Dexon sutures (Dexon, B. Braun-Dexon, marked LNCaP luciferase-neomycin tumor cells ortho- Spangenberg, Germany), and the skin was again disin- topically implanted into the prostates of SCID mice.9 fected using a Dibromol tincture (Trommsdorf GmbH, Moreover, to optimize sensitivity, we used high lucifer- Alsdorf, Germany). ase-expressing LNCaP LLN cell pools, which were generated by transduction using a VSV-G-pseudotyped lentivirus. Measurement of in vivo bioluminescence Animals were injected with 100 mlofD-luciferin (Synchem OHG, Felsberg/Altenburg, Germany) and anesthetized Generation of luciferase-expressing LNCaP LLN cells using isofluorane. Ten minutes after the injection, The luciferase–neomycin fusion gene cassette was images of mice were acquired using a NIGHTOWL 17,18 inserted into a lentiviral backbone vector, using camera (Berthold Technologies, Bad Wildbad, Germany) BsRGi/SnaBI replacing the original expression cassette. equipped with isofluorane adaptors. An overview The 293T cells were transfected with this lentiviral picture of the mice and 2 exposures, 5 min at 10 Â 10 backbone and plasmids pCMV VSV-G (encoding the binning and 1 min at 2 Â 2 binning, were taken. The vesicular stomatitis virus surface gene) and pCMV 8–74 intensity of the emitted light was quantified over (GAG/REV) using Ca–phosphate precipitates. Forty- the whole animal using the software provided with the eight and seventy-two hours later, viral supernatants NIGHTOWL CCD camera (Berthold Technologies) and were harvested, filtered through 0.4-mM filters and added expressed as photons per second. to LNCaP cells together with 4 mgmlÀ1 polybrene. The supernatants were left for ca. 8 h and exchanged for normal medium. Transduced LNCaP LLN cells were Necropsy and luciferase assays of mouse organs selected using 3 mg mlÀ1 G418 for 1 week and used as a At the end of the experiment, mice were killed, the cell pool. This protocol yielded cell pools with B10 times abdominal cavity was opened and a picture of the tumor higher levels of luciferase activity than the previously in situ was taken. The primary tumor was resected, weighed, reported retroviral vector (LNCaP luciferase-neomycin: measured and its volume was calculated using the formula ca. 800 LU (light units) per cell versus LNCaP LLN: ca. V ¼a2 Â A/2 (a: small diameter, A: large diameter). 10 000 LU per cell). Tumors were subsequently homogenized in a luciferase lysis buffer. To obtain a quantitative analysis of the metastatic spread into potential target organs, the lung, Orientating toxicity studies liver, inguinal lymph nodes and lumbar spine, as the To estimate the maximum tolerated dose of our novel most common metastatic sites in prostate cancer, were prodrug PSA9, we intravenously injected 4 groups of resected from the animals taking great care to prevent healthy male C.B-17/SCID mice (3 animals each) with 6, 8, À1 cross-contamination of the tissues. After homogeniza- 12 and 24 mg kg doxorubicin equivalents of PSA9 as the tion, the insoluble material was removed by centrifuga- albumin-binding prodrug. The doses were injected on tion for 10 min at 3000 r.p.m. in a Heraeus Megafuge 2.0 days 1, 8 and 15. Body weight, as an indicator for drug (Thermo Scientific, Karlsruhe, Germany). In all, 5 ml was toxicity, was recorded three times per week. On day 24, the checked for protein concentration using a Bradford assay toxicity study was terminated and animals were killed. (Sigma B6916, Sigma, Seelze, Germany) with bovine serum albumin serving as a standard protein and 10 ml In vivo animal experiment was measured in a luciferase assay (Promega E4550, Male SCID (C.B-17/Icr-HanHsd-Prkdc-scid) mice ran- Promega, Mannheim, Germany). ging between 6 and 8 weeks of age (approximate weight, 30 g) were housed under controlled conditions. They were fed a diet consisting of not o20% proteins, 5% Results and Discussion fibers, 3.5% fats and 6.5% ashes and supplied with vitamins mixture and water ad libitum and exposed Orientating toxicity studies revealed that a noticeable to 12-h light and dark cycle before experiments. The weight loss of 420% between day 1 and day 8 was À1 animals were maintained in separated conventional observed in animals treated with 12 and 24 mg kg housing at constant temperature and humidity and kept doxorubicin equivalents of PSA9. In addition, one animal in the animal house for 2 weeks for adaptation. of each group was found dead on day 8. Therefore, treatment with these two doses was stopped. Treatment with 8 mg kgÀ1 doxorubicin equivalents of PSA9 admi- Animal model design nistered as the albumin-binding prodrug also induced a Mice were anesthetized with isofluorane, positioned and weight loss of B20% between day 1 and day 10. In fixed on the back. The coat of the abdominal area was addition, one of the three animals in this group was shaved and the skin was disinfected with 70% alcohol. A found dead on day 12. On the other hand, no significant 3–4 mm incision was made alongside the linea alba in the weight loss was observed in the group treated with À lower abdomen using the cranial edge of the bladder for 6mgkg 1 doxorubicin equivalents of PSA9, and all the orientation. Seminal vesicles and prostate were pulled three animals of this group survived without any signs of out partially and exposed carefully. In all, 2 Â 106 tumor toxicity up to the end of the experiment (day 24).

Prostate Cancer and Prostatic Diseases Prodrug of doxorubicin B Elsadek et al 17 Table 1 Dose schedule, mortality and body weight loss of 110 doxorubicin- and PSA9-treated groups in comparison with the control untreated group in the orthotopic PSA-positive LNCaP model in vivo Substance SCID Treatment Dosea (mg kgÀ1 Toxic BWC (%) 100 mice (d) per injection) i.v. deaths d 25–44

5% Glucose 8 25, 32, 39 2 B14 buffer Doxorubicin 8 25, 32, 39 3 1 B18 90 PSA9 8 25, 32, 39 6 0 B12 start of treatment Abbreviations: BWC, body weight change; d, days; i.v., intravenous; SCID, (day 25) severe combined immunodeficiency. aDoxorubicin equivalents. 80

Vehicle (n=6) animal weight (% original weight) Treatment of SCID mice orthotopically implanted with Doxorubicin (n=7; 3x3mg/kg) PSA9 (n=8; 3x6mg/kg) LNCaP LLN cells 70 À1 On preliminary toxicity studies, 3 Â 6mgkg doxo- 0 5 10 15 20 25 30 35 40 45 rubicin equivalents of PSA9 were compared with day 3 Â 3mgkgÀ1 of free doxorubicin in a dose schedule illustrated in Table 1. 2.5x1009 The doses of doxorubicin were lower than those used Vehicle (n=6) in the previously reported experiment for PSA5 Doxorubicin (n=7; 3x3mg/kg) (2 Â 4mgkgÀ1)9 as 2 of the 10 treated animals died in PSA9 (n=8; 3x6mg/kg) 2.0x1009 this group and body weight loss exceeded 20%. The maximum tolerated dose of PSA9 over PSA5 was two- fold lower, which is very likely because of the improved 1.5x1009 cleavage profile of PSA9 that effectively releases active start of treatment doxorubicin as the final cleavage product. A group (day 25) receiving 5% glucose solution served as a negative 09 control. 1.0x10 Ten days after orthotopic implantation of LNCaP LLN cells, the in vivo growth of tumor cells was first measured 08 using bioluminescence (BLI). Another 14 days later, mice 5.0x10 in vivo luciferase activity (ph/s) were randomized into treatment groups according to their in vivo BLI signals, and mice without detectable tumors were excluded from the study. The take rate was 0.0 B60%, underlining the importance of using high 0 5 10 15 20 25 30 35 40 45 luciferase-expressing LNCaP LLN cells and BLI for day ensuring that only tumor-bearing animals are included Figure 2 (a) Body weight change (% of original weight) from the in the in vivo study. Both body weights and clinical beginning up to the end of the experiment in the in vivo LNCaP symptoms of animals were recorded three times weekly. study with PSA9. (b) In vivo growth curves as measured by After 3 weeks of treatment (44 days after implantation) bioluminescence during treatment with doxorubicin and PSA9. For animals were killed, and tumor sizes and weights were luciferase in vivo measurements, animals were injected with 100 ml of D-luciferin, anesthetized, and 10 min after the injection, images of measured. Furthermore, a part of the primary tumors as mice were acquired using a NIGHTOWL camera. Light emission well as lung, liver, inguinal lymph nodes and lumbar over the whole animal was quantified as photons per second using spine as potential target organs for metastases were the Winlight Software (Berthold Technologies). harvested, homogenized and assayed for luciferase activity as a measure for tumor cell content. Finally, blood was collected from the animals, and serum PSA levels and treatment may have been the cause in the case of were measured with a nephelometric immunoassay from doxorubicin. Abbott (Wiesbaden, Germany) as a luciferase-indepen- BLI measurements of the luciferase-expressing LNCaP dent biomarker for total tumor cell quantification. LLN cells were used to assess the growth of the tumor As shown in Figure 2a, animals of all three groups lost once weekly. The in vivo BLI activity of the treated weight during the experiment. The weight loss started groups was higher than that of the control group before treatment, suggesting primary tumor burden or (Figure 2b). The most likely reason for these differences the metastatic spread of the tumor cells rather than the is attributed to the above-mentioned deaths of the two treatment as a possible cause (see supporting informa- animals in the control group and one in the doxorubicin- tion). Following the onset of treatment on day 25, the treated group (see Table 1), which most likely reduced decrease in body weights was most pronounced in the the total average tumor sizes in these groups. In addition, group treated with doxorubicin, and one animal died in with increasing tumor sizes, the linearity of the BLI this group on day 25. For the control group, in which two signal decreases because of an increase in necrotic areas animals died during the course of the experiment, and a saturation of the signal. tumor-related problems were the most likely cause for In contrast to in vivo BLI measurements, callipered their demise, whereas a combination of tumor burden tumor sizes and the luciferase assay at the end of the

Prostate Cancer and Prostatic Diseases Prodrug of doxorubicin B Elsadek et al

18 200 Vehicle Doxorubicin (3x3mg/kg) 175 PSA9 (3x6mg/kg)

150 n=7 n=6 125 n=6 n=7 100 n=8

75 n=8

50 percent of mean control

25 p=0.27 p=0.1 p=0.09 0 tumor volumetumor luciferase PSA level

300 Vehicle (n=6) Vehicle (n=8) Doxorubicin (n=7; 3x3mg/kg) Doxorubicin (n=8; 2x4mg/kg) 250 PSA9 (n=8; 3x6mg/kg) PSA5 (n=8; 3x12mg/kg) $ = Graeser et al., 2008

200

150 p=0.56 100 p=0.38

percent of mean control 50 **p=0.0098

$$$*p=0.024 $$$ $$$ $$ $ 0 lung metastases *bone metastaseslymph node metastases liver metastases Figure 3 The antitumor and antimetastatic activity of PSA9 compared with doxorubicin and an untreated control group and the previous data with PSA5 in the orthotopic PSA-positive model (LNCaP).9 The mean of the control group was set at 100%, and the other values were calculated accordingly. Students’ t-test was used for statistical analysis. (a) The antitumor efficacy of PSA9 as measured by callipering, tumor volume, luciferase activity and PSA serum levels. (b) Antimetastatic activities of PSA9 (bold colors) on lung, bone, lymph-node and liver metastases compared with the results from the previously reported PSA5 results, including the respective controls (light colors).9 In this study, the lumbar spine was assayed for luciferase activity; in the previous study, the femur served as an example for bone metastases. The apparent pro-tumor and pro-metastatic effect of doxorubicin in the results from the current study must be noted, which very likely reflects the loss of two animals in the control group. It is also noteworthy that in the PSA5 study, both doxorubicin and PSA5 showed some but not statistically significant efficacy against lung metastases, but no activity against any other metastatic site.9

experiment showed an increased antitumor activity of organs of control animals compared with the previous PSA9 at 3 Â 6mgkgÀ1 doxorubicin equivalents over the study, thus resulting in a more stable readout. Therapy doxorubicin-treated and control groups—see Table 1 and with PSA9 produced statistically significantly reduced Figure 3a (P ¼ 0.27 (tumor size) and P ¼ 0.1 (luciferase luciferase activities in the lung and lumbar spine when activity) versus control). compared with control animals (see Figure 3b). More- The most likely explanation for the apparent tumor over, PSA9 showed antimetastatic effects against both growth-stimulating activity of doxorubicin is given above, hepatic and lymph-node metastases, although these were the reduction of the control group by two animals that not statistically significant. Doxorubicin treatment, on died during the experiment (see Figure 3a). Last but not the other hand, did not reduce the spread of tumor cells least, PSA serum levels confirmed the antitumor/anti- to any of these organs (Figure 3b). metastatic effect of PSA9 with the lowest levels compared Conversely, our previously designed PSA5 prodrug, with doxorubicin and close to statistical significance which lacks the PABC linker, showed moderate activity versus the control group (P ¼ 0.09, Figure 3a). against lung metastases, which was comparable with doxorubicin at 2 Â 4mgkgÀ1, but neither treatment showed any efficacy against any other metastases (see Figure 3b). The clearly improved antimetastatic efficacy Effects on lung, liver, inguinal lymph nodes and lumbar of PSA9 is an encouraging result, considering that the spine metastases major cause of death in prostate cancer patients is Luciferase assays of potential target organs are a means metastatic disease.1,2 to quantify metastatic tumor cells and analyze the effect Thereasonswhysomemetastaticsitesweremorerespon- of antimetastatic treatment. The use of the high luciferase- sive to PSA9 could be that the first two metastatic sites (the expressing LNCaP LLN cell pool generated in this study lung and lumbar spine) are major targets for metastatic yielded B10 times higher luciferase activities in the LNCaP cells, and therefore have a relatively high average

Prostate Cancer and Prostatic Diseases Prodrug of doxorubicin B Elsadek et al 19

Figure 4 A comparison of in vivo luciferase with integrisense-mediated 3D fluorescence molecular tomography imaging of orthotopically implanted LNCaP lentiviral luciferase-neomycin cells. (a) In vivo luciferase imaging (2 Â 2 binning, 1 min) pictures. The massive signal emanating from the lower abdomen, that is, the area of the prostate must be noted. No signal was detected from the lung area, also with 10 Â 10 binning and 5 min exposure (data not shown). (b) Fluorescence imaging 3 days after integrisense injection: The main graph displays a scan of the area of the primary tumor (blue square). As we did not detect any specific signal from the upper area in the analyzed animals, the animals were opened and rescanned. Only one animal gave a detectable signal in the area of the lung (green circle), which was, however, too weak for a 3D reconstruction. The three white dots are nonspecific signals from the front paws and the snout of the animals. A strong signal emanating from the primary tumors could be detected in vivo and ex vivo (orange circles). The color reproduction of this figure is available on the html full text version of the manuscript. luciferase activity (14 000 and 13 000 LU per mgprotein).The mediated in vivo fluorescence imaging. However, lymph nodes and the liver (8000 and 4000 LU per mgprotein, whereas the primary tumor was readily detected in vivo, respectively) are secondary sites only, with a more variable lung metastases could only be seen on opening the metastasizingprofile.Thus,themorestablesignalsfromthe animal’s chest, and only in the animal, which displayed first two sites in the control group allow for a more robust the highest in vitro luciferase activity (Figures 4a and b). comparison between control and treatment. The size of the The quantification of the in vivo three-dimensional metastases and varying levels of proteases that cleave the fluorescence imaging for the primary tumor correlated prodrugs and/or the dipeptide in the different metastases well with results from the in vivo and in vitro luciferase arealsoapossibleexplanation. activities and with the actual tumor size (see Table 2). Besides achieving more stable in vitro luciferase assay Thus, both sensitive in vivo tumor measurement mod- results, we were hoping that the increase in luciferase alities were in good agreement regarding the detection of activity per cell would enable the detection of signals the primary tumor, but our data indicate that more sensi- from lung metastases in vivo. However, despite consis- tive imaging probes, for example, imaging compounds tently high in vitro luciferase activities from the lungs of that exploit the quenching effects of tumor-susceptible tumor-bearing control animals, no corresponding signal probes, are required to image metastases effectively.21 could be detected in vivo. One problem might be the In summary, incorporating the self-eliminating PABC disperse distribution of the numerous, but small metas- spacer between the peptide sequence and doxorubicin tases in the lung, which will generate a rather scattered improved the cleavage profile liberating free doxorubicin light signal compared with the primary tumor or larger, effectively in the PSA-positive tumor tissue. This well-defined metastases. In addition, the difference improvement is translated preclinically in vivo in the between the luciferase activities of the primary versus form of a promising antitumor efficacy of PSA9 using all the lung metastases is quite large, which requires a broad modes of necropsy analyses (namely tumor size, lucifer- dynamic range to display both signals at the same time. ase activity and PSA serum levels), although just below Recently, an alternative very sensitive method to detect significance versus nontreated animals. In contrast, the tumors in vivo was developed using an avb3 integrin- clinically established doxorubicin showed no effect at all binding fluorescent probe (‘integrisense’) in combination (Figure 3a). Furthermore, luciferase assays as a mean to with a fluorescence molecular tomographic imaging measure tumor cell spreading into organs for metastatic system.19,20 Therefore, three animals that had not been disease, demonstrated that PSA9, but not the free drug, included in the study were subjected to integrisense- was able to significantly decrease the metastatic spread

Prostate Cancer and Prostatic Diseases Prodrug of doxorubicin B Elsadek et al 20 Table 2 Quantification of in vivo and in vitro BLI and integrisense fluorescence of three tumor-bearing mice (LNCaP) Mice code Tumor Metastases

In vivo In vitro Volume (cm3) Lung

Luciferase Integrisense Luciferase Integrisense Luciferase Integrisense (photons/s) (pmol) (LU) (count/energy) (LU) (count/energy)

87 5.79E+08 48.01 428 040 531.47 0.0875 10 407 — 107 1.13E+09 101.94 1 071 370 790.05 0.512 1 296 080 93.66 138 1.99E+08 64.45 368 520 259.86 0.144 1 060 340 —

Abbreviation: BLI, bioluminescence; FMT, fluorescence molecular tomography. Three mice that were not included in the main study were subjected to in vivo BLI before they were injected with integrisense 750 probe (2.4 nmol per mouse). Images were quantified using NIGHTOWL (Berthold Technologies; BLI) or a Visen FMT 1500 instrument, Visen Medical, Bedford, MA, USA (fluorescence). Animals were then killed, opened and subjected again to fluorescence imaging (‘in vitro’/metastases lung). Tumors and lungs were then removed, homogenized and their luciferase activity determined (‘in vitro’/metastases lung).

of LNCaP LLN cells to the lung, liver, inguinal lymph Doxorubicin HSA9 nodes and the lumbar spine. Compared with the 0.4 previous study with the PSA-cleavable prodrug PSA5, the antitumor efficacy of PSA9 on the primary tumor was comparable but the antimetastatic activity of PSA9 was 0.3 considerably improved. When considering the loss of 24 h two animals of the control group in this study, the results 0.2 for PSA9 seem even more impressive and indicate a clear 17 h therapeutic benefit over PSA5. In the clinical setting, PSA levels in human primary 0.1 2 h prostate tumors can be very high depending on the stage 0 h

of the disease (up to 1600–2100 nM) and 80–90% of the Absorbance (mV) 495 nm 0.0 secreted PSA is active in the tumor environment.22 PSA is secreted in an active form in prostate cancer but forms 0 5 10 15 20 25 30 35 40 45 50 Minutes two stable complexes with a1-antichymotrypsin and a2-macroglobulin in the blood. PSA complexed to a1-antichymotrypsin is the predominant fraction of Doxorubicin HSA9 PSA. Only a minor fraction of serum PSA is not 0.4 associated with proteinase inhibitors, and it is unknown 23 whether this free fraction still has enzymatic activity. In 0.3 addition, it has recently been shown that PSA in mice bearing LNCaP tumors forms complexes similar to those 24 h 0.2 in humans but the major immunoreactive complex 16 h contains a1-antitrypsin rather than a1-antichymotryp- 23 sin. To ensure that our novel prodrug PSA9 was not 0.1 2 h being cleaved prematurely in the serum, we incubated the albumin conjugate of PSA9 with murine serum from 0 h the experiment in the LNCaP model that contains a high Absorbance (mV) at 495 nm 0.0 PSA serum level from a control tumor-bearing animal 0505 10 15 20 25 30 35 40 45 B À1 (PSA 150 ng ml ) and with human serum from a patient Minutes with prostate cancer that showed a very high level of circulating PSA in his serum (PSA B718 ng mlÀ1). As Figure 5 Stability studies of the albumin conjugate of PSA9 (HSA9—100 mM)in(a) murine serum from mouse with PSA- shown in Figures 5a and b, only marginal cleavage positive LNCaP prostate cancer (PSA serum level B150 ng mlÀ1) of doxorubicin or other by-products were observed over and in (b) human serum of a patient with prostate cancer (PSA 24 h at 37 1C in either case. serum level B718 ng mlÀ1). Reversed-phase HPLC studies were performed on a BioLogic Duo-Flow System (Munich, Germany) using a solvent delivery system (BioLogic Duo-Flow 1082), an internal UV detector (l ¼ 280 nm), an external UV detector, Bischoff Lambda 1010 (l ¼ 495 nm), a Nucleosil C18-column (100-5, Conclusions 250 Â 4mm2, Macherey-Nagel, Du¨ ren, Germany) and Bio-Rad software (Munich, Germany) for data analysis; HPLC conditions: In this study, we developed an albumin-binding prodrug flow rate: 1 ml per min; mobile phase A: 20% MeCN, 80% water and of doxorubicin (PSA9) incorporating a PSA substrate that 0.1% HCl; mobile phase B: 50% MeCN, 50% water and 0.1% HCl: is cleaved selectively by PSA releasing doxorubicin gradient: 0–5 min 100% mobile phase A; 5–35 min increase to 100% efficiently as the final cleavage product due to the mobile phase B; 35–40 min 100% mobile phase B; 40–45 min decrease to 100% mobile phase A; 45–50 min 100% mobile phase incorporation of PABC as a self-immolative spacer. The A; injection volume: 50 ml. prodrug PSA9 demonstrated superior efficacy over doxorubicin regarding the tumor growth of the primary metastases is noteworthy, considering that such metas- tumor and on the spread of metastases. The pronounced tases are often responsible for the poor quality life of and statistically significant effect on lung and bone prostate cancer patients and mortality. In this sense,

Prostate Cancer and Prostatic Diseases Prodrug of doxorubicin B Elsadek et al 21 PSA9 is an optimized prodrug over PSA5 that did not 4 Armstrong AJ, Garrett-Mayer ES, Yang YC, de Wit R, Tannock IF, release doxorubicin and showed less activity regarding Eisenberger M. A contemporary prognostic nomogram for men the development of metastases. with hormone-refractory metastatic prostate cancer: a TAX327 Nevertheless, further preclinical development of PSA- study analysis. Clin Cancer Res 2007; 13: 6396–6403. cleavable prodrugs based on PSA9 as a lead structure is 5 Armstrong AJ, Carducci MA. Chemotherapy for advanced necessary to identify a candidate for clinical develop- prostate cancer: results of new clinical trials and future studies. Curr Oncol Rep 2005; 7: 220–227. ment. Considering that the primary prostate tumor grows 6 Calabro F, Sternberg CN. Current indications for chemotherapy slowly and that the spread of metastases generally occurs in prostate cancer patients. Eur Urol 2007; 51: 17–26. over a long period of time, a subsequent step in our 7 Denmeade SR, Nagy A, Gao J, Lilja H, Schally AV, Isaacs JT. preclinical development to further improve the efficacy of Enzymatic activation of a doxorubicin-peptide prodrug by the outlined prodrug strategy for treating prostate cancer prostate-specific antigen. Cancer Res 1998; 58: 2537–2540. would be to investigate an improved long-acting dosing 8 DeFeo-Jones D, Garsky VM, Wong BK, Feng DM, Bolyar T, Haskell schedule with our doxorubicin prodrug using a parent- K et al. A peptide-doxorubicin ‘prodrug’ activated by prostate- eral route, as prostate tumors in the majority of cases specific antigen selectively kills prostate tumor cells positive for grow over a long time span, and a three- or four-weekly prostate-specific antigen in vivo. Nat Med 2000; 6: 1248–1252. dose scheme with conventional anticancer agents, which 9 Graeser R, Chung DE, Esser N, Moor S, Schachtele C, Unger C et al. Synthesis and biological evaluation of an albumin-binding only reaches high anticancer drug peaks in the prostate prodrug of doxorubicin that is cleaved by prostate-specific tumor for a very short period, is in our opinion not an antigen (PSA) in a PSA-positive orthotopic prostate carcinoma optimal way of treating slow-growing tumors. Therefore, model (LNCaP). Int J Cancer 2008; 122: 1145–1154. another innovative approach that we are currently 10 Kratz F. Albumin as a drug carrier: design of prodrugs, drug investigating in our group is the development of orally conjugates and nanoparticles. J Control Release 2008; 132: 171–183. applicable albumin-binding prodrugs. Administration of 11 Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular such agents bearing a PSA-cleavable substrate as a long- permeability and the EPR effect in macromolecular therapeutics: term oral dosage form should ideally allow a continuous a review. J Control Release 2000; 65: 271–284. and critical concentration of the drug to be released in the 12 Kratz F, Warnecke A, Schmid B, Chung DE, Gitzel M. Prodrugs prostate primary tumor and in metastases over a long of anthracyclines in cancer chemotherapy. Curr Med Chem 2006; 13: 477–523. time span, thus reducing systemic toxicity and optimiz- 13 Kratz F. DOXO-EMCH (INNO-206): the first albumin-binding ing the therapeutic outcome of our prodrug approach. prodrug of doxorubicin to enter clinical trials. Expert Opin Such an approach, although certainly ambitious, could Investig Drugs 2007; 16: 855–866. transfer favorably to tumors that grow slowly such as 14 Elsadek B, Graeser R, Warnecke A, Unger C, Saleem T, prostate tumors and to achieve long-term stabilization or El-Melegy N et al. Optimization of an albumin-binding prodrug reduction in tumor growth. The development of orally of doxorubicin that is cleaved by prostate-specific antigen (PSA). applicable albumin prodrugs with PSA cleavage seems ACS Med Chem Lett 2010; 1: 234–238. feasible and worthy of further investigation to obtain a 15 Carl PL, Chakravarty PK, Katzenellenbogen JA. A novel critical cytotoxic concentration over a long and constant connector linkage applicable in prodrug design. J Med Chem time span within the heterogeneous prostate tumor 1981; 24: 479–480. characterized by tumor cells in different cell cycles, 16 Workman P, Balmain A, Hickman JA, McNally NJ, Rohas AM, Mitchison NA et al. UKCCCR guidelines for the welfare of which allows the patient to dose an oral prodrug animals in experimental neoplasia. Lab Anim 1988; 22: 195–201. according to his personal needs. 17 Demaison C, Parsley K, Brouns G, Scherr M, Battmer K, Kinnon C et al. High-level transduction and gene expression in hemato- poietic repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus type 1-based lentiviral Conflict of interest vector containing an internal spleen focus forming virus The authors declare no conflict of interest. promoter. Hum Gene Ther 2002; 13: 803–813. 18 Zhang F, Thornhill SI, Howe SJ, Ulaganathan M, Schambach A, Sinclair J et al. Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide Acknowledgements highly reproducible and stable transgene expression in hemato- poietic cells. Blood 2007; 110: 1448–1457. The financial supports of the Dietmar Hopp Foundation, 19 Bornmann C, Graeser R, Esser N, Ziroli V, Jantscheff P, Keck T Germany, and the Cultural Affairs and Missions Sector, et al. A new liposomal formulation of Gemcitabine is active in an Egyptian Ministry of Higher Education are gratefully orthotopic mouse model of pancreatic cancer accessible to acknowledged. bioluminescence imaging. Cancer Chemother Pharmacol 2008; 61: 395–405. 20 Kossodo S, Pickarski M, Lin SA, Gleason A, Gaspar R, Buono C et al. Dual in vivo quantification of integrin-targeted and References protease-activated agents in cancer using fluorescence molecular tomography (FMT). Mol Imaging Biol 2010; 12: 488–499. 1 Jaeger EB, Samant RS, Rinker-Schaeffer CW. Metastasis suppres- 21 Hilderbrand SA, Weissleder R. Near-infrared fluorescence: sion in prostate cancer. Cancer Metastasis Rev 2001; 20: 279–286. application to in vivo molecular imaging. Curr Opin Chem Biol 2 Serretta V, Altieri V, Morgia G, Siragusa A, De Grande G, Napoli M 2010; 14: 71–79. et al. Oral chemotherapy in hormone-refractory prostate carcinoma 22 Denmeade SR, Sokoll LJ, Chan DW, Khan SR, Isaacs JT. patients unwilling to be admitted to hospital. Urol Int 2009; 83: Concentration of enzymatically active prostate-specific antigen 452–457. (PSA) in the extracellular fluid of primary human prostate cancers 3 Arlen PM, Gulley JL. Docetaxel-based regimens, the standard of and human prostate cancer xenograft models. Prostate 2001; 48: 1–6. care for metastatic androgen-insensitive prostate cancer. Future 23 Ravery V, Boccon-Gibod L. Free/total prostate-specific antigen Oncol 2005; 1: 19–22. ratio—hope and controversies. Eur Urol 1997; 31: 385–388.

Prostate Cancer and Prostatic Diseases