IJO International Journal of Oncology 1019-6439 1791-2423 D.A. Spandidos 10.3892/ijo.2012.1754 ijo-42-02-0373 Review Cathepsin B-cleavable doxorubicin prodrugs for targeted cancer therapy ZHONGYAN-JUN12 SHAOLI-HUA12 LIYAN12 Department of Oncology, Zhongnan Hospital of Wuhan University; Wuhan 430071, P.R. China Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, Wuhan 430071, P.R. China Correspondence to: Professor Yan Li, Department of Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu road, Wuhan 430071, P.R. China, E-mail: liyansd2@163.com 2 2013 28 12 2012 42 2 373 383 25 10 2012 03 12 2012 Copyright © 2013, Spandidos Publications 2013 This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

Doxorubicin (DOX) is one of the most effective cytotoxic anticancer drugs used for the treatment of hematological malignancies, as well as a broad range of solid tumors. However, the clinical applications of this drug have long been limited due to its severe dose-dependent toxicities. Therefore, DOX derivatives and analogs have been developed to address this issue. A type of DOX prodrug, cleaved by cathepsin B (Cat B), which is highly upregulated in malignant tumors and premalignant lesions, has been developed to achieve a higher DOX concentration in tumor tissue and a lower concentration in normal tissue, so as to enhance the efficacy and reduce toxicity to normal cells. In this review, we focused on Cat B-cleavable DOX prodrugs and discussed the efficacy of these prodrugs, demonstrated by preclinical and clinical developments.

 cancer chemotherapy cathepsin B doxorubicin prodrugs targeted therapy Contents

Introduction

Prodrug strategies in cancer treatment

Cathepsin B (Cat B) as a prodrug-activating enzyme

Cat B-cleavable DOX prodrugs

Conclusions

 Introduction

Chemotherapy is a major therapeutic approach for the treatment of cancer. Doxorubicin (DOX; Fig. 1), an anthracycline isolated from Streptomyces strains, is one of the most effective anticancer drugs used for the treatment of hematological malignancies and a broad range of solid tumors, including lymphoma, Kaposi’s sarcoma, bone tumors, as well as stomach, breast and ovarian cancers (1,2). DOX in its salt form is readily distributed into almost all tissues and intracellular compartments via passive diffusion or active transport following intravenous administration, resulting in indiscriminative toxic effects on all cells exposed to it. Therefore, the clinical application of DOX is limited by its dose-dependent side-effects, such as bone marrow toxicity, cardiotoxicity, nephrotoxicity and hepatotoxicity.

To reduce the side-effects of this drug, significant efforts have been made to develop DOX derivatives and analogs with less toxic effects and improved pharmacological properties. Several strategies have been investigated in clinical and preclinical trials, including various methods of administration, combinations with other chemotherapeutic drugs [e.g., adriamycin, bleomycin, vinblastine and dacarbazine (ABVD), cyclophosphamide, hydroxydaunomycin, oncovin and prednisone (CHOP)] (3), the addition of antioxidant nutrients (4) and cardioprotectors (5–7), the development of liposomes (8) and nanoparticles (9), the effects of acute exercise (10) and the development of prodrugs (11–13). In this review, we focused on the DOX prodrug strategies.

 Prodrug strategies in cancer treatment

Prodrugs are derivatives of drugs which remain inactive in their prototype form but are metabolized in the body to generate the active drugs at the site of action. They are particularly useful in the development of novel antitumor chemotherapeutic drugs, leading to reduced toxicity, improved specificity and the avoidance of multidrug resistance (14,15). The use of prodrugs for targeted therapy is usually based on tumor-associated cell surface markers, such as antigens or receptors, whose expression differs between normal and cancer cells (16,17). Several prodrug strategies have been pursued, including active and passive targeting approaches with antibodies, serum proteins, liposomes and synthetic polymers (18–22). There have been some classic and clinically successful prodrugs, such as capecitabine, an enzyme-activated prodrug, which is converted into 5-fluoro uridine or 5-fluoro-2-deoxyuridine in tumor cells to achieve targeted cytotoxicity (23).

Prodrugs can be divided into high- and low-molecular weight drugs in terms of molecular weight (Mw). The former are internalized by passive or active endocytosis and ultimately become localized in the lysosomal components of cells, while the latter usually enter cells mainly by diffusion (24). The Mw and biodistribution of drugs have important impacts on antitumor efficacy. Macromolecular drugs accumulate in tumor tissues due to the enhanced permeability and retention effect (25–27). A Mw below the renal threshold (∼50,000 g/mol) is rapidly lost from the circulation; therefore, macro-molecular weight drugs may have increased intravascular half-lives, resulting in an increased therapeutic efficacy (27). N-(2-hydroxypropyl) methacrylamide (HPMA), known as one of the most widely used prototypic polymeric drug carriers, was first used to synthesize polymeric drugs in the 1970s, due to its non-immunogenic and non-toxic properties and long circulating half-life (28,29). It has been demonstrated that an HPMA-copolymer Mw of 200,000 to 600,000 g/mol is desirable for the efficient passive targeting of solid tumors (30). Prodrugs bearing HPMA have been developed in preclinical studies and include caplostatin (31,32), P-GDM (33,34) and P-HYD-IgG (35), as well as in phase I/II clinical studies and included HPMA copolymer-Gly-Phe-Leu-Gly-doxorubicin (PK1) (36–39), galactosamine-targeted poly(HPMA)-doxorubicin (PK2) (40–42), PK3 (36), PNU166945 (43), AP5346 (44–48) and AP5280 (49–51).

 Cathepsin B (Cat B) as a prodrug-activating enzyme

Some tumor-associated enzymes, such as proteases, glucuronidases or carboxylesterases, expressed intra- or extracellularly in cancer cells, can release or activate prodrugs. Cat B, a lysosomal cysteine protease in normal cells and tissues, is considered to be one of the best examples of intracellular proteases. It is highly upregulated in malignant tumors and premalignant lesions at the mRNA and protein levels (52). Cat B is localized in perinuclear vesicles, presumably lysosomes in normal cells. However, in tumor cells and oncogene-transformed cells, Cat B is localized in perinuclear vesicles and vesicles throughout the cytoplasm and at the cell periphery (53). Pericellular Cat B participates in degrading processes associated with tumor proliferation, invasion and metastasis. Moreover, exposure to DOX can induce a time- and dose-dependent upregulation of Cat B expression at the mRNA and protein levels (5).

Cat B cleaves Leu, Arg-Arg, Ala-Leu, Phe-Arg, Phe-Lys, Ala-Phe-Lys, Gly-Leu-Phe-Gly, Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu (18,54–58). There are several low- and high-Mw DOX prodrugs that can be activated by Cat B. Furthermore, DOX immunoconjugates, in which DOX is linked to a carcinoma-specific antibody through Cat B-cleavable oligopeptides, have also been designed (59). All of these conjugates have shown rapid and almost quantitative DOX release in the presence of Cat B. The rate of DOX release depends on the length and structure of the spacer. The tetrapeptide, Gly-Phe-Leu-Gly, has been found to be one of the most suitable spacers. In this regard, the steric interaction between the peptide substrate and Cat B has a significant impact on the release of DOX from prodrugs (60). Therefore, to decrease the steric interaction, it is necessary to integrate a self-immolative spacer, such as para-aminobenzyloxycarbonyl (PABC) between the drug and the oligopeptide substrate.

 Cat B-cleavable DOX prodrugs

Examples of Cat B-cleavable DOX prodrugs are illustrated in Fig. 2 and summarized in Table I.

DOX prodrugs containing the tetrapeptide Gly-Phe-Leu-Gly

The tetrapeptide, Gly-Phe-Leu-Gly, has been proven to be the most effective with respect to both plasma stability and rapid hydrolysis in the presence of Cat B. Therefore, many DOX prodrugs are based on this tetrapeptide.

 PK1

PK1 [FCE28068; P(GFLG)-ADR; DOX-HPMA; doxorubicin-HPMA copolymer conjugate; HPMA-doxorubicin, 8 wt% DOX; Fig. 3A], a polymeric prodrug of Mw ∼30,000 g/mol, was the first macromolecular prodrug to enter clinical trials, and has reached phase II clinical trials.

Preclinical studies using tumor cells, including L1210 leukemia (61–64), A2780 and DOX-resistant A2780/AD ovarian carcinoma cells, have shown that PK1 can partially avoid the ATP-driven P-glycoprotein (Pgp) efflux pump compared with free DOX (65–67). The IC50 doses of free DOX and PK1 account for the differences in the mechanisms of cellular uptake (65). In preclinical studies using animal models, including B16F10 melanoma, L1210 leukemia, M5076, LS174T human colorectal xenografts (64) and sensitive and resistant human ovarian carcinoma models (68), PK1 has shown enhanced efficacy. The release of DOX from PK1 in vitro and in vivo using HPLC analysis has shown only a single peak, representing DOX (64). PK1 does not release DOX in the plasma and the covalently-bound drug is biologically inactive following intravenous administration.

Phase I clinical studies on patients with solid tumors, including colorectal, breast, biliary tract, pancreatic, urinary tract, head/neck, non-small cell lung (NSCL), mesothelioma and stomach cancers, have shown that the maximum tolerated dose (MTD) for PK1 is 320 mg/m2, which is 4- to 5-fold higher than the usual clinical dose of free DOX (60–80 mg/m2) (69). PK1 decreases non-specific organ toxicities by several folds and allows the active drug to be delivered intracellularly, while maintaining antitumor activity (36,39). Phase II studies using PK1 have shown decreased toxicity with evident activity in breast, NSCL and colorectal cancers. Furthermore, SPECT and γ-camera imaging with 123I-labelled drugs have shown obvious tumor accumulation in two metastatic breast cancers (38). PK1 and free DOX greatly differ in their antiproliferative effects and cell death signals in EL-4 cancer cells; treatment with free DOX greatly increases p38 phosphorylation, while PK1 increases it only slightly; PK1 also significantly increases ERK phosphorylation, while free DOX slightly decreases it (70).

In addition, polymer-directed enzyme prodrug therapy (PDEPT) combining HPMA copolymer-Cat B and PK1 has shown activity against a COR-L23 xenograft, whereas PK1 alone has not and in B16F10 melanoma tumors PDEPT has been shown to more effective than either PK1 or free DOX alone (71).

 PK2

PK2 (FCE28069, 27,000 g/mol, 8 wt% DOX; Fig. 3B), the only targeted polymer conjugate containing galactosamine to enter clinical trials, is designed to target the asialoglycoprotein receptor (ASGPR) which is selectively expressed in hepatocytes and hepatoma cell lines (40,72). Pharmacokinetic studies using PK2 in rats and mice have shown effective liver targeting with >70% of the released DOX being selectively targeted to the liver following intravenous administration (73,74). Preclinical studies using rats have demonstrated that PK2 displays a ∼5-fold reduction in cardiotoxicity as opposed to free DOX following intravenous or intraperitoneal administration at various doses (11,64). Furthermore, antitumor activity has been shown to be improved in rodent tumor models (69).

In a pivotal study on a patient with multifocal hepato-cellular carcinoma, HPLC and 123I-based imaging showed the biphasic clearance of PK2 from the plasma (half-life, 78±1 and 990±15 min) and ∼30% of the delivered drug accumulated in the liver at 24 h. Moreover, SPECT analysis showed that the radioactivity concentration was 3- to 4-fold higher in peritu-moral liver tissue than in the tumor tissue itself (40).

Phase I/II trials have shown that the MTD of PK2 is 160 mg/m2 (DOX equivalent) and several hepatocellular carcinoma patients have displayed partial responses and/or stable disease (41). γ-camera imaging and CT scanning have revealed that 15–20% of total PK2 is retained in the liver and is mostly concentrated in normal liver tissue (normal versus tumor tissue, 5:1), suggesting that the galactosamine-targeted polymer is mainly delivered to normal regions of the liver due to the increased ASGPR expression in the normal liver (75) and the phagocytosis by Kupffer cells with ‘galactose particle’ receptor expression (76). Despite this disparity in PK2 distribution, the drug concentration in tumor tissue was still 12- to 50-fold higher than it would have been with the administration of free DOX alone.

 HPMA copolymer-doxorubicin conjugates (P-DOX)

P-DOX conjugates (Fig. 3C) (77,78) contain the oligopeptide Gly-Phe-Leu-Gly and the N2,N5-bis(N-methacryloyl-glycylphenylalanyl-leucyl-glycyl) ornithine cross-linker, which permits the synthesis of P-DOX conjugates with various Mws, from 22 to 1230 kDa. The clearance rate of P-DOX from the blood is Mw-dependent and is much slower than that of free DOX (26,77). The therapeutic efficacy has been shown to increase as the Mw of P-DOX increases in nude mice bearing subcutaneous OVCAR-3 xenografts. The low residual concentration of P-DOX in tissues (apart from tumors) helps to avoid potential long-term side-effects (77). The toxicity against hematopoietic precursors and normal lymphocytes of inbred mice is considerably decreased (78).

 HPMA copolymer-DOX-OV-TL16 [P-(GFLG)-DOX-Ab]

P-(GFLG)-DOX-Ab (270,000 g/mol, 3.3 wt% DOX; Fig. 4A) is recognized by the OA3 antigen, which plays a role in membrane transport and/or signal transduction for its multimembrane-spanning domain structure (59,79–81). The P-(GFLG)-DOX-Ab is rapidly absorbed by OVCAR-3 cells and transported into their lysosomal compartment. DOX is subsequently released from the conjugate at the site with a degradable GFLG spacer, diffused via the lysosomal membrane and accumulates in the cell nuclei (80). Preliminary data on the relative retention of DOX in MDR (A2780/AD) cells have indicated a higher intracellular DOX concentration after incubation with HPMA copolymer-DOX conjugate compared with free DOX (59).

 HPMA copolymer-Gly-Phe-Lys-Gly-DOX-N-acylated galactosamine [P-(GFLG)-DOX-GalN]

P-(GFLG)-DOX-GalN (25,000 g/mol; Fig. 4B), contains N-acylated galactosamine (GalN), which was designed to be recognized by ASGPR in HepG2 human hepatocellular carcinoma cells (59,82) and individual members of the galectin family (e.g., galectin-3) in human colon adenocarcinoma (83,84). Galectin-3 is expressed in normal tissues and highly expressed in neoplastic tissues (85–87); although the exact opposite has been shown to occur (88,89). In SW-480 and SW-620 cells, the presence of galectin-3 on the cell surface has been demonstrated by flow cytometry; however, it has not been detected on the surface of Colo-205 cells. The cellular cytotoxicity of P-(GFLG)-DOXGalN determined by MTT assay has been shown to be ∼10-fold higher than P-GFLG-DOX and 10-fold higher in Colo-205 cells than in SW-480 and SW-620 cells (90). This suggests the participation of other galectins, such as galectin-1, -4, -7 or -8, in P-(GFLG)-DOX-GalN targeting.

 Lactose-containing HPMA copolymer-doxorubicin conjugate [P-(GFLG-DOX)-lac]

P-(GFLG-DOX)-lac (Fig. 4C) (90), can also be biorecognized by galectin-3 on the surface of colon cancer cells. The in vitro cytoxicity determined by MTT assay is higher than that of the non-glycosylated P-(GFLG)-DOX product and almost 1,000-fold lower than that of free DOX in HepG2 human hepatocellular carcinoma cells and Colo-205, SW-480 and SW-620 colon adenocarcinoma cells.

 Trivalent galactose-containing HPMA copolymerdoxorubicin conjugate [P-(GFLG-DOX)-TriGal]

P-(GFLG-DOX)-TriGal (Fig. 4D) contains trivalent galactose, which can also be biorecognized by galectin-3 on the surface of colon adenocarcinoma cells. The cytotoxicity of the P-(GFLGDOX)-TriGal has been shown to be at least 10-fold higher than that of the non-glycosylated P-(GFLG)-DOX product in Colo-205, SW-480 and SW-620 colon adenocarcinoma cells (90).

 N-Methacryloyl-glycyl)-dl-phenylalanyl-leucyl-glycyl-DOX (Ma-GFLG-DOX)

Ma-GFLG-DOX contains the tetrapeptide, Gly-Phe-Leu-Gly (91,92). It remains quite stable in buffer at pH 7.4 (model of the bloodstream), but releases DOX either under mild acidic conditions or in the presence of Cat B (rich in the tumor microenvironment).

 PAMAM dendrimers (D-NH2)-Gly-Phe-Leu-Gly-HPMA-doxorubicin (D2-GFLG-P-DOX)

D2-GFLG-P-DOX (215,000 g/mol, 9.2 wt% DOX), which is attached to DOX via a pH-sensitive hydrazone bond (91,93), was prepared by grafting the semitelechelic HPMA copolymers, which have Mws below the renal threshold, onto a PAMAM dendrimer core via a biodegradable linkage GFLG oligopeptide. An in vitro study using phosphate buffers at pH 5.0 or 7.4 at 37°C (hydrazone conjugates) and in a Cat B-containing (5×10−7 M) phosphate buffer at 37°C (amide conjugates) showed that the presence of Cat B increased the rate of DOX release (91).

 HMW1D

HMW1D (115,000 g/mol, 7.4 wt% DOX), a branched polymer prodrug, contains water-soluble polymer drug carriers, HPMA copolymers, and a biodegradable oligopeptide sequence, GFLG, linking shorter polymer chains (Mw, 20,000 g/mol) into a high-Mw structure (Mw, 110,000 g/mol) to enhance the passive accumulation of the drug by increasing its Mw. An in vitro study showed that this pH-sensitive prodrug (HMW1D) can be degraded by Cat B (5×10−7 M), 37°C, pH 6.0 (93).

 TET1D

TET1D (19,600 g/mol, 10.5 wt% DOX; Fig. 5) (93) a non-targeted polymer-bound doxorubicin conjugate, contains a hydrazone bond, which significantly improves the rate of DOX release, compared with that of classical HPMA polymer prodrugs bearing DOX attached via amide bonds limited to maximum 8–9 wt%. An in vitro study using T-splenocytes and mouse EL-4 T cell lymphoma cells showed that the toxicity of TET1D is much higher compared with that of similar classic conjugates and an in vivo study using EL4 T cell lymphoma mice C57BL/10 showed that the antitumor activity was also significantly increased. An in vitro study showed that TET1D can be cleaved by Cat B; however, Cat B is not essential in the release of DOX, for it also contains a pH-sensitive spacer which is stable under physiological conditions (pH 7.4, e.g., blood) and hydrolytically degradable in a mild acidic environment (pH 5.0, e.g., endosome) (93).

 DOX prodrugs containing the tetrapeptide, Ala-Leu-Ala-Leu 6-Maleimidocaproic acid-Arg-Arg-Ala-Leu-Ala-Leu-DOX (EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOX)

EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOX bears maleimide (94), which can rapidly and selectively react in situ with the cysteine-34 position of circulating albumin after intravenous administration and release the drug at the tumor site (95,96). Albumin is a promising drug carrier due to its passive accumulation in solid tumors, which have a high metabolic turnover, angiogenesis, hypervasculature, defective vascular architecture and impaired lymphatic drainage (97). Albumin has non-toxic, non-immunogenic, biocompatible and biodegradable properties (98) and has demonstrated preferential tumor uptake in various tumor xenograft animal models (99). The antitumor efficacy of EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOX has been shown to be comparable to that of free DOX in a M-3366 breast cancer xenograft model at equivalent doses (94). Moreover, the albumin-binding DOX prodrug, DOX-EMCH (INNO-206), has been examined in clinical trials (100,101).

 DOX prodrugs containing the dipeptide, Phe-Lys Ac-Phe-Lys-PABC-DOX

Ac-Phe-Lys-PABC-DOX (PDOX, 1045.5 g/mol, 52.0% DOX, Fig. 6A) contains the dipeptide, Phe-Lys, which is specific for Cat B and the self-immolative spacer, PABC (12,102–104). An in vivo study using a nude mice model of gastric cancer with peritoneal carcinomatosis showed that, compared with free DOX, PDOX (16 mg/kg, twice that of DOX in terms of equal molecular content) produced better antitumor effects in terms of experimental peritoneal carcinomatosis index (ePCI) (Fig. 7A) and body weight (Fig. 7D), and reduced liver (Fig. 7B and C), kidney (Fig. 7E and F) and heart (Fig. 7G–I) toxicities (12).

 ε-maleimidocaproic acid-Phe-Lys-PABC-DOX (EMC-Phe-Lys-PABC-DOX)

EMC-Phe-Lys-PABC-DOX (Fig. 6B) (2,18,104) has exhibited dramatic differences in antitumor activity between in vitro and in vivo studies. An in vitro cytotoxicity study using the pancreatic tumor cell line, AsPC1 LN, and the melanoma cancer cell line, MDA-MB-231 LN, showed that DOX was ∼6-fold more active than the prodrug. However, an in vivo study using a breast cancer xeno-graft nude mice model of MDA-MB-435 cells showed that the prodrug exhibited superior antitumor activity (tumor size, 15% of that in nude mice treated with the vehicle) compared to DOX (tumor size, 49% of that in nude mice treated with the vehicle) in an equitoxic comparison (2).

 PG-Phe-Lys-DOX

Hyperbranched polyglycerol-Phe-Lys-DOX (PG-Phe-Lys-DOX, 45% DOX) (18,41,105), contains the dipeptide, Phe-Lys, and hyperbranched polyglycerol. The drug release of the conjugates suggested an effective cleavage of PG-Phe-Lys-DOX and release of DOX in the presence of Cat B. The IC50 of PG-Phe-Lys-DOX in the breast cancer cell line, MDA-MB-231, and the pancreatic carcinoma cell line, AsPC1, was 1.10±0.4 and 2.4±0.6 μM, respectively, both of which were lower than that of free DOX (105).

 Z-Phe-Lys-PABC-DOX

Benzyloxycarbonyl-Phe-Lys-PABC-DOX (Z-Phe-Lys-PABC-DOX; Fig. 6C), is stable in human plasma and rapidly releases DOX in the presence of Cat B at 37°C, pH 5.0 (half-life, 8 min), which is 30-fold faster than that of the Val-Cit conjugate. On the other hand, the release rate is significantly faster than Z-Phe-Lys-DOX, suggesting that a self immolative spacer, such as PABC, is helpful for DOX release from conjugates (104).

 BR96-SC-Phe-Lys-PABC-DOX

BR96-SC-Phe-Lys-PABC-DOX (Fig. 6D) contains the chimeric monoclonal antibody, BR96, that binds specifically to a Lewisy-related, tumor-associated antigen expressed on the surface of many human carcinoma cells. An in vitro study using human carcinomal cell lines expressing varying levels of the BR96 antigen showed that the cytotoxicity of BR96-Phe-Lys-PABC-DOX was directly related to the level of antigen expression on the cell membrane: the higher level of BR96 antigen, the higher the sensitivity to BR96-Phe-Lys-PABC-DOX. The cytotoxicity of BR96-Phe-Lys-PABC-DOX in high BR96 antigen-expressing cell lines is higher than that of the non-binding IgG-SC-Phe-Lys-PABC-DOX conjugate (>220-fold), confirming its BR96 antigen specificity (104).

 Other DOX prodrugs containing dipeptides

Dubowchik et al(104) and de Groot et al(106) synthesized a series of other DOX prodrugs containing the dipeptides, Phe-Lys, Ala-Lys or Phe-Arg, including Z-Phe-Lys-PABC-DOX•HCl, MC-Phe-Lys(MMT)-PABC-DOX, MC-Phe-Lys-PABC-DOX•Cl2CHCO2H, Z-Phe-Lys(alloc)-DOX, Z-Phe-Lys-DOX•HCl, Z-Ala-Lys(alloc)-PABC-DOX, Z-Ala-Lys-PABC-DOX•HCl, Z-Phe-Arg(NO2)-PABC-DOX, Z-Phe-Arg(Ts)-PABC-DOX, Fmoc-Phe-Lys(Aloc)-PABC-DOX and H-Phe-Lys(Aloc)-PABC-DOX. However, data regarding their antitumor activity are lacking.

 Conclusions

Over the past few decades, significant efforts have been made to develop antitumor prodrugs with increased efficacy and decreased toxicity. Numerous DOX prodrugs have been synthesized by structure modification strategies. Cat B-cleavable DOX prodrugs release the free drugs in the presence of Cat B and in a subacidic environment. A number of in vitro cancer cell studies and in vivo tumor xenograft studies have demonstrated Cat B-cleavable DOX prodrugs to be less toxic in vitro and more effective in vivo, demonstrating the role of Cat B.

However, there remain many challenges and questions. The majority of the studies mentioned in this review are in a very early preclinical stage with little information on physicochemical properties, cytotoxicity and antitumor efficacy in tumor cells and xenografts. The subcellular distribution of the prodrugs, the free drugs released and the antitumor mechanisms remain unclear. Further studies are warranted and should focus on preclinical and clinical evaluation of existing prodrugs, rather than synthesizing novel drug candidates in this field.

 Abbreviations: Cat B

cathepsin B

 DOX

doxorubicin

 HPMA

N-(2-hydroxypropyl)methacrylamide

 PK1

HPMA copolymer-Gly-Phe-Leu-Gly-doxorubicin

 PK2

galactosamine-targeted poly(HPMA)-doxorubicin

 P-DOX

HPMA copolymer-doxorubicin conjugates

 P-(GFLG)-DOX-Ab

HPMA copolymer-DOX-OV-TL16

 P-(GFLG)-DOX-GalN

HPMA copolymer-Gly-Phe-Lys-Gly-DOX-N-acylated galactosamine

 P-(GFLG-DOX)-lac

lactose-containing HPMA copolymer-doxorubicin conjugate

 P-(GFLG-DOX)-TriGal

trivalent galactose-containing HPMA copolymer-doxorubicin conjugate

 Ma-GFLG-DOX

(N-methacryloyl-glycyl)-dl-phenylalanyl-leucylglycyl-DOX

 D2-GFLG-P-DOX

PAMAM dendrimers (D-NH2)-Gly-Phe-Leu-Gly-HPMA-doxorubicin

 EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOX

6-maleimidocaproic acid-Arg-Arg-Ala-Leu-Ala-Leu-DOX

 EMC-Phe-Lys-PABC-DOX

ε-maleimidocaproic acid-Phe-Lys-PABC-DOX

 PG-Phe-Lys-DOX

hyperbranched polyglycerol-Phe-Lys-DOX

 Z-Phe-Lys-PABC-DOX

benzyloxycarbonyl-Phe-Lys-PABC-DOX

This study was supported by the State Key Research Project on Infectious Diseases (2012ZX10002012-012) and the National Natural Science Foundation of China (no. 81171396) and National University Students Innovation Training Project of China (101048639).

 References GianniLGrasselliGCrestaSLocatelliAViganoLMinottiGAnthracyclinesCancer Chemother Biol Response Modif2129402003 Abu AjajKGraeserRFichtnerIKratzFIn vitro and in vivo study of an albumin-binding prodrug of doxorubicin that is cleaved by cathepsin BCancer Chemother Pharmacol644134182009 OguraMAdriamycin (doxorubicin)Gan To Kagaku Ryoho28133113382001(In Japanese). Granados-PrincipalSQuilesJLRamirez-TortosaCLSanchez-RoviraPRamirez-TortosaMCNew advances in molecular mechanisms and the prevention of adriamycin toxicity by antioxidant nutrientsFood Chem Toxicol48142514382010 HermanEHFerransVJJordanWArdalanBReduction of chronic daunorubicin cardiotoxicity by ICRF-187 in rabbitsRes Commun Chem Pathol Pharmacol3185971981 WexlerLHAndrichMPVenzonDRandomized trial of the cardioprotective agent ICRF-187 in pediatric sarcoma patients treated with doxorubicinJ Clin Oncol143623721996 LipshultzSEDexrazoxane for protection against cardiotoxic effects of anthracyclines in childrenJ Clin Oncol143283311996 CattelLCerutiMDosioFFrom conventional to stealth liposomes: a new frontier in cancer chemotherapyTumori892372492003 LiJWuCDaiYZhangRWangXFuDChenBDoxorubicin-CdS nanoparticles: a potential anticancer agent for enhancing the drug uptake of cancer cellsJ Nanosci Nanotechnol74354392007 AscensaoALumini-OliveiraJMachadoNGAcute exercise protects against calcium-induced cardiac mitochondrial permeability transition pore opening in doxorubicin-treated ratsClin Sci12037492011 YeungTKHopewellJWSimmondsRHReduced cardiotoxicity of doxorubicin given in the form of N-(2-hydroxypropyl) methacrylamide conjugates: and experimental study in the ratCancer Chemother Pharmacol291051111991 ShaoLHLiuSPHouJXCathepsin B cleavable novel prodrug Ac-Phe-Lys-PABC-ADM enhances efficacy at reduced toxicity in treating gastric cancer peritoneal carcinomatosis: an experimental studyCancer118298629962011 KratzFWarneckeASchmidBChungDEGitzelMProdrugs of anthracyclines in cancer chemotherapyCurr Med Chem134775232006 MullerMBKeckMEBinderEBABCB1 (MDR1)-type P-glycoproteins at the blood-brain barrier modulate the activity of the hypothalamic-pituitary-adrenocortical system: implications for affective disorderNeuropsychopharmacology28199119992003 GottesmanMMFojoTBatesSEMultidrug resistance in cancer: role of ATP-dependent transportersNat Rev Cancer248582002 LuYYangJSegaEIssues related to targeted delivery of proteins and peptidesAAPS J8E466E4782006 Juillerat-JeanneretLSchmittFChemical modification of therapeutic drugs or drug vector systems to achieve targeted therapy: looking for the grailMed Res Rev275745902007 CalderonMGraeserRKratzFHaagRDevelopment of enzymatically cleavable prodrugs derived from dendritic polyglycerolBioorg Med Chem Lett19372537282009 HaagRKratzFPolymer therapeutics: concepts and applicationsAngew Chem Int Ed Engl45119812152006 DuncanRPolymer conjugates as anticancer nanomedicinesNat Rev Cancer66887012006 VicentMJDieudonneLCarbajoRJPineda-LucenaAPolymer conjugates as therapeutics: future trends, challenges and opportunitiesExpert Opin Drug Deliv55936142008 KiickKLMaterials science. Polymer therapeuticsScience317118211832007 SchilskyRLPharmacology and clinical status of capecitabineOncology1412971306discussion 1309–1311, 2000. BasuSKReceptor-mediated endocytosis of macromolecular conjugates in selective drug deliveryBiochem Pharmacol40194119461990 NoguchiYWuJDuncanRStrohalmJUlbrichKAkaikeTMaedaHEarly phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissuesJpn J Cancer Res893073141998 ShiahJJSunYPetersonCMKopecekJBiodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorin e(6) and adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenograftsJ Control Release611451571999 BogdanovAJrWrightSCMarecosEMBogdanovaAMartinCPetherickPWeisslederRA long-circulating co-polymer in ‘passive targeting’ to solid tumorsJ Drug Target43213301997 KopecekJSprinclLLimDNew types of synthetic infusion solutions. I. Investigation of the effect of solutions of some hydrophilic polymers on bloodJ Biomed Mater Res71791911973 SprinclLExnerJSterbaOKopecekJNew types of synthetic infusion solutions. III. Elimination and retention of poly-[N-(2-hydroxypropyl)methacrylamide] in a test organismJ Biomed Mater Res109539631976 EtrychTKovarLStrohalmJChytilPRihovaBUlbrichKBiodegradable star HPMA polymer-drug conjugates: biodegradability, distribution and anti-tumor efficacyJ Control Release1542412482011 Satchi-FainaroRPuderMDaviesJWTargeting angio-genesis with a conjugate of HPMA copolymer and TNP-470Nat Med102552612004 Satchi-FainaroRMamlukRWangLInhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatinCancer Cell72512612005 KasuyaYLuZRKopeckovaPMinkoTTabibiSEKopecekJSynthesis and characterization of HPMA copolymeraminopropylgeldanamycin conjugatesJ Control Release742032112001 NishiyamaNNoriAMaluginAKasuyaYKopeckovaPKopecekJFree and N-(2-hydroxypropyl)methacrylamide copolymer-bound geldanamycin derivative induce different stress responses in A2780 human ovarian carcinoma cellsCancer Res63787678822003 EtrychTMrkvanTRihovaBUlbrichKStar-shaped immunoglobulin-containing HPMA-based conjugates with doxorubicin for cancer therapyJ Control Release12231382007 VaseyPAKayeSBMorrisonRPhase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: first member of a new class of chemo-therapeutic agents-drug-polymer conjugates. Cancer Research Campaign Phase I/II CommitteeClin Cancer Res583941999 BilimVTechnology evaluation: PK1, Pfizer/Cancer Research UKCurr Opin Mol Ther53263302003 SeymourLWFerryDRKerrDJPhase II studies of polymer-doxorubicin (PK1, FCE28068) in the treatment of breast, lung and colorectal cancerInt J Oncol34162916362009 ThomsonAHVaseyPAMurrayLSCassidyJFraierDFrigerioETwelvesCPopulation pharmacokinetics in phase I drug development: a phase I study of PK1 in patients with solid tumoursBr J Cancer81991071999 JulyanPJSeymourLWFerryDRPreliminary clinical study of the distribution of HPMA copolymers bearing doxorubicin and galactosamineJ Control Release572812901999 SeymourLWFerryDRAndersonDHepatic drug targeting: phase I evaluation of polymer-bound doxorubicinJ Clin Oncol20166816762002 SeymourLWUlbrichKWedgeSRHumeICStrohalmJDuncanRN-(2-hydroxypropyl)methacrylamide copolymers targeted to the hepatocyte galactose-receptor: pharmacokinetics in DBA2 miceBr J Cancer638598661991 Meerum TerwogtJMten Bokkel HuininkWWSchellensJHPhase I clinical and pharmacokinetic study of PNU166945, a novel water-soluble polymer-conjugated prodrug of paclitaxelAnticancer Drugs123153232001 RiceJRGerberichJLNowotnikDPHowellSBPreclinical efficacy and pharmacokinetics of AP5346, a novel diaminocyclohexane-platinum tumor-targeting drug delivery systemClin Cancer Res12224822542006 NowotnikDPCvitkovicEProLindac (AP5346): a review of the development of an HPMA DACH platinum polymer therapeuticAdv Drug Deliv Rev61121412192009 CamponeMRademaker-LakhaiJMBennounaJHowellSBNowotnikDPBeijnenJHSchellensJHPhase I and pharmacokinetic trial of AP5346, a DACH-platinum-polymer conjugate, administered weekly for three out of every 4 weeks to advanced solid tumor patientsCancer Chemother Pharmacol605235332007 Van der SchootSCNuijenBSoodPThurmondKBIIStewartDRRiceJRBeijnenJHPharmaceutical development, quality control, stability and compatibility of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum antineoplastic agent AP5346Pharmazie618358442006 SoodPThurmondKBIIJacobJEWallerLKSilvaGOStewartDRNowotnikDPSynthesis and characterization of AP5346, a novel polymer-linked diaminocyclohexyl platinum chemotherapeutic agentBioconjug Chem17127012792006 Rademaker-LakhaiJMTerretCHowellSBA Phase I and pharmacological study of the platinum polymer AP5280 given as an intravenous infusion once every 3 weeks in patients with solid tumorsClin Cancer Res10338633952004 TibbenMMRademaker-LakhaiJMRiceJRStewartDRSchellensJHBeijnenJHDetermination of total platinum in plasma and plasma ultrafiltrate, from subjects dosed with the platinum-containing N-(2-hydroxypropyl)methacrylamide copolymer AP5280, by use of graphite-furnace Zeeman atomic-absorption spectrometryAnal Bioanal Chem3732332362002 LinXZhangQRiceJRStewartDRNowotnikDPHowellSBImproved targeting of platinum chemotherapeutics. the antitumour activity of the HPMA copolymer platinum agent AP5280 in murine tumour modelsEur J Cancer402912972004 PodgorskiISloaneBFCathepsin B and its role(s) in cancer progressionBiochem Soc Symp702632762003 CalkinsCCSameniMKoblinskiJSloaneBFMoinKDifferential localization of cysteine protease inhibitors and a target cysteine protease, cathepsin B, by immuno-confocal microscopyJ Histochem Cytochem467457511998 KovarMStrohalmJEtrychTUlbrichKRihovaBStar structure of antibody-targeted HPMA copolymer-bound doxorubicin: a novel type of polymeric conjugate for targeted drug delivery with potent antitumor effectBioconjug Chem132062152002 ThanouMDuncanRPolymer-protein and polymer-drug conjugates in cancer therapyCurr Opin Investig Drugs47017092003 MaiJWaismanDMSloaneBFCell surface complex of cathepsin B/annexin II tetramer in malignant progressionBiochim Biophys Acta14772152302000 KratzFMullerIARyppaCWarneckeAProdrug strategies in anticancer chemotherapyChemMedChem320532008 TrouetAMasquelierMBaurainRDeprez-De CampeneereDA covalent linkage between daunorubicin and proteins that is stable in serum and reversible by lysosomal hydro-lases, as required for a lysosomotropic drug-carrier conjugate: in vitro and in vivo studiesProc Natl Acad Sci USA796266291982 OmelyanenkoVKopeckovaPGentryCKopecekJTargetable HPMA copolymer-adriamycin conjugates. Recognition, internalization and subcellular fateJ Control Release5325371998 CarlPLChakravartyPKKatzenellenbogenJAA novel connector linkage applicable in prodrug designJ Med Chem244794801981 SeymourLWUlbrichKSteygerPSBreretonMSubrVStrohalmJDuncanRTumour tropism and anti-cancer efficacy of polymer-based doxorubicin prodrugs in the treatment of subcutaneous murine B16F10 melanomaBr J Cancer706366411994 DuncanRKopeckovaPStrohalmJHumeICLloydJBKopecekJAnticancer agents coupled to N-(2-hydroxypropyl) methacrylamide copolymers. II. Evaluation of daunomycin conjugates in vivo against L1210 leukaemiaBr J Cancer571471561988 DuncanRKopeckova-RejmanovaPStrohalmJAnticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitroBr J Cancer551651741987 HopewelJWDuncanRWildingDChakrabartiKPreclinical evaluation of the cardiotoxicity of PK2: a novel HPMA copolymer-doxorubicin-galactosamine conjugate antitumour agentHum Exp Toxicol204614702001 MinkoTKopeckovaPPozharovVKopecekJHPMA copolymer bound adriamycin overcomes MDR1 gene encoded resistance in a human ovarian carcinoma cell lineJ Control Release542232331998 MinkoTKopeckovaPKopecekJChronic exposure to HPMA copolymer-bound adriamycin does not induce multidrug resistance in a human ovarian carcinoma cell lineJ Control Release591331481999 TijerinaMFowersKDKopeckovaPKopecekJChronic exposure of human ovarian carcinoma cells to free or HPMA copolymer-bound mesochlorin e6 does not induce P-glycoprotein-mediated multidrug resistanceBiomaterials21220322102000 MinkoTKopeckovaPKopecekJEfficacy of the chemo-therapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinomaInt J Cancer861081172000 DuncanRDrug-polymer conjugates: potential for improved chemotherapyAnticancer Drugs31752101992 KovarLStrohalmJChytilPThe same drug but a different mechanism of action: comparison of free doxorubicin with two different N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugates in EL-4 cancer cell lineBioconjug Chem188949022007 SatchiRConnorsTADuncanRPDEPT: polymer-directed enzyme prodrug therapy. I. HPMA copolymer-cathepsin B and PK1 as a model combinationBr J Cancer85107010762001 PaulAVicentMJDuncanRUsing small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugatesBiomacromolecules8157315792007 PimmMVPerkinsACStrohalmJUlbrichKDuncanRGamma scintigraphy of a 123I-labelled N-(2-hydroxypropyl) methacrylamide copolymer-doxorubicin conjugate containing galactosamine following intravenous administration to nude mice bearing hepatic human colon carcinomaJ Drug Target33853901996 DuncanRSeymourLCScarlettLLloydJBRejmanovaPKopecekJFate of N-(2-hydroxypropyl)methacrylamide copolymers with pendent galactosamine residues after intravenous administration to ratsBiochim Biophys Acta88062711986 VirgoliniIMullerCKlepetkoWAngelbergerPBergmannHO’GradyJSinzingerHDecreased hepatic function in patients with hepatoma or liver metastasis monitored by a hepatocyte specific galactosylated radioligandBr J Cancer619379411990 Schlepper-SchaferJHulsmannDDjovkarAMeyerHEHerbertzLKolbHKolb-BachofenVEndocytosis via galactose receptors in vivo. Ligand size directs uptake by hepatocytes and/or liver macrophagesExp Cell Res1654945061986 ShiahJGDvorakMKopeckovaPSunYPetersonCMKopecekJBiodistribution and antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates in nude miceEur J Cancer371311392001 RihovaBBilejMVetvickaVUlbrichKStrohalmJKopecekJDuncanRBiocompatibility of N-(2-hydroxypropyl) methacrylamide copolymers containing adriamycin. Immunogenicity and effect on haematopoietic stem cells in bone marrow in vivo and mouse splenocytes and human peripheral blood lymphocytes in vitroBiomaterials103353421989 OmelyanenkoVKopeckovaPGentryCShiahJGKopecekJHPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. 1. influence of the method of synthesis on the binding affinity to OVCAR-3 ovarian carcinoma cells in vitroJ Drug Target33573731996 OmelyanenkoVGentryCKopeckovaPKopecekJHPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. II. Processing in epithelial ovarian carcinoma cells in vitroInt J Cancer756006081998 KunathKKopeckovaPMinkoTKopecekJHPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. 3. The effect of free and polymer-bound adriamycin on the expression of some genes in the OVCAR-3 human ovarian carcinoma cell lineEur J Pharm Biopharm4911152000 JensenKDKopeckovaPBridgeJHKopecekJThe cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in Hep G2 cellsAAPS PharmSci3E322001 DavidAKopeckovaPKopecekJRubinsteinAThe role of galactose, lactose and galactose valency in the biorecognition of N-(2-hydroxypropyl)methacrylamide copolymers by human colon adenocarcinoma cellsPharm Res19111411222002 DavidAKopeckovaPRubinsteinAKopecekJEnhanced biorecognition and internalization of HPMA copolymers containing multiple or multivalent carbohydrate side-chains by human hepatocarcinoma cellsBioconjug Chem128908992001 IrimuraTMatsushitaYSuttonRCIncreased content of an endogenous lactose-binding lectin in human colorectal carcinoma progressed to metastatic stagesCancer Res513873931991 BresalierRSMazurekNSternbergLRByrdJCYunkerCKNangia-MakkerPRazAMetastasis of human colon cancer is altered by modifying expression of the beta-galactoside-binding protein galectin 3Gastroenterology1152872961998 OhannesianDWLotanDThomasPJessupJMFukudaMGabiusHJLotanRCarcinoembryonic antigen and other glycoconjugates act as ligands for galectin-3 in human colon carcinoma cellsCancer Res55219121991995 LotzMMAndrewsCWJrKorzeliusCALeeECSteeleGDJrClarkeAMercurioAMDecreased expression of Mac-2 (carbohydrate binding protein 35) and loss of its nuclear localization are associated with the neoplastic progression of colon carcinomaProc Natl Acad Sci USA90346634701993 CastronovoVCampoEvan den BruleFAInverse modulation of steady-state messenger RNA levels of two non-integrin laminin-binding proteins in human colon carcinomaJ Natl Cancer Inst84116111691992 DavidAKopeckovaPMinkoTRubinsteinAKopecekJDesign of a multivalent galactoside ligand for selective targeting of HPMA copolymer-doxorubicin conjugates to human colon cancer cellsEur J Cancer401481572004 EtrychTStrohalmJChytilPCernochPStarovoytovaLPecharMUlbrichKBiodegradable star HPMA polymer conjugates of doxorubicin for passive tumor targetingEur J Pharm Sci425275392011 DvorakMKopeckovaPKopecekJHigh-molecular weight HPMA copolymer-adriamycin conjugatesJ Control Release603213321999 EtrychTJelinkovaMRihovaBUlbrichKNew HPMA copolymers containing doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological propertiesJ Control Release73891022001 SchmidBChungDEWarneckeAFichtnerIKratzFAlbumin-binding prodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker that are cleaved by cathepsin B: synthesis and antitumor efficacyBioconjug Chem187027162007 KratzFWarneckeAScheuermannKProbing the cysteine-34 position of endogenous serum albumin with thiol-binding doxorubicin derivatives. Improved efficacy of an acid-sensitive doxorubicin derivative with specific albumin-binding properties compared to that of the parent compoundJ Med Chem45552355332002 WarneckeAKratzFMaleimide-oligo(ethylene glycol) derivatives of camptothecin as albumin-binding prodrugs: synthesis and antitumor efficacyBioconjug Chem143773872003 KratzFBeyerUSerum proteins as drug carriers of anti-cancer agents: a reviewDrug Deliv52812991998 ElzoghbyAOSamyWMElgindyNAAlbumin-based nanoparticles as potential controlled release drug delivery systemsJ Control Release1571681822012 KratzFAlbumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticlesJ Control Release1321711832008 LebrechtDGeistAKetelsenUPHaberstrohJSetzerBKratzFWalkerUAThe 6-maleimidocaproyl hydrazone derivative of doxorubicin (DOXO-EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damageInt J Cancer1209279342007 UngerCHaringBMedingerMDrevsJSteinbildSKratzFMrossKPhase I and pharmacokinetic study of the (6-maleimidocaproyl)hydrazone derivative of doxorubicinClin Cancer Res13485848662007 DubowchikGMFirestoneRACathepsin B-sensitive dipeptide prodrugs. 1. A model study of structural requirements for efficient release of doxorubicinBioorg Med Chem Lett8334133461998 DubowchikGMMosureKKnipeJOFirestoneRACathepsin B-sensitive dipeptide prodrugs. 2. Models of anti-cancer drugs paclitaxel (Taxol), mitomycin C and doxorubicinBioorg Med Chem Lett8334733521998 DubowchikGMFirestoneRAPadillaLCathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activityBioconjug Chem138558692002 CalderónMQuadirMAStrumiaMHaagRFunctional dendritic polymer architectures as stimuli-responsive nano-carriersBiochimie92124212512010 De GrootFMBroxtermanHJAdamsHPDesign, synthesis and biological evaluation of a dual tumor-specific motive containing integrin-targeted plasmin-cleavable doxorubicin prodrugMol Cancer Ther19019112002 Figures and Table

Sructure of DOX. DOX contains an amino group (-NH2) on the sixmembered ring, which can conjugate with a carboxyl group (-COOH), and a carbonyl group (-C═O) on another six-membered ring which can react with amino groups. These are the two most common conjugating sites for prodrug design.

Examples of DOX prodrugs. Macromolecular DOX prodrugs are conjugated with polymer carriers, oligopeptides, with or without antibodies, which can combine with antigens located on the tumor cell surface and ligands that can be recognized by receptors on the tumor cell surface. Low molecular weight DOX prodrugs are combined with oligopeptides, with or without antibodies and ligands. DOX prodrugs remain inactive in blood vessels and normal tissues, but are cleaved by Cat B in tumor tissues and tumor cells, releasing free DOX, which results in targeted cytotoxicity. DOX, doxorubicin; Cat B, cathepsin B.

General chemical structures of (A) PK1, (B) PK2 and (C) P-DOX, in which DOX is complexed with (2-hydroxypropyl) methacrylamide (HPMA), by the tetrapeptide linker, Gly-Phe-Leu-Gly (GFLG).

General chemical structures of (A) P-(GFLG)-DOX-Ab, (B) P-(GFLG)-DOX-GalN, (C) P-(GFLG)-DOX-lac and (D) P-(GFLG)-DOX-TriGal, in which DOX is conjugated with HPMA, by the tetrapeptide linker, Gly-Phe-Leu-Gly (GFLG).

Structure of TET1D, in which DOX is combined with the tetrapep-tide, Gly-Phe-Leu-Gly (GFLG).

Structure of (A) Ac-Phe-Lys-PABC-DOX, (B) EMC-Phe-Lys-PABC-DOX, (C) Z-Phe-Lys-PABC-DOX and (D) BR96-SC-Phe-Lys-PABC-DOX, in which DOX is linked to the self-immolative spacer, para-aminobenzyloxycarbonyl (PABC) and the dipeptide, Phe-Lys (FL).

Cat B-cleavable prodrug Ac-Phe-Lys-PABC-DOX (PDOX) enhances treatment efficacy and reduces toxicity in treating gastric cancer with peritoneal carcinomatosis [modified from a previous study (12)]. (A) Effects of DOX and PDOX on a peritoneal carcinomatosis model are shown with the detailed experimental peritoneal carcinomatosis index (ePCI) score; both DOX and PDOX significantly reduced the ePCI. PDOX reduced general toxicity and toxicity to the liver, kidney and the heart in particular. (D) Nude mice in the PDOX group had similar body weights to those in the control group throughout the study period, while nude mice in the DOX group showed a progressive decrease in body weight after 4 doses of intraperitoneal injection. Effects of PDOX and DOX on major liver and renal function parameters are shown in (B) ALT, (C) AST, (E) BUN and (F) Cr. PDOX significantly decreased hepatotoxicity compared with DOX in terms of AST. PDOX significantly decreased myocardial toxicity compared with DOX by reducing (G) CK, (H) CK-MB and (I) LDH. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine; CK, creatine kinase; CK-MB, creatine kinase-MB isoenzyme; LDH, lactate dehydrogenase.

List of Cat B-cleavable DOX prodrugs.

Name Biodegradable spacer Mw (g/mol) DOX proportion Current status MTD Refs.
DOX None 543.5 100% Clinical therapy 60–80 mg/m2 (69)
PK1 Gly-Phe-Leu-Gly 30,000 8 (wt%) Phase II 320 mg/m2 (36,38,39, 61–71)
PK2 Gly-Phe-Leu-Gly 27,000 8 (wt%) Phase I/II 160 mg/m2 (10,11,40, 41,72–76)
P-DOX Gly-Phe-Leu-Gly 22,000–1,230,000 NA Preclinical ND 26,77,78
P-(GFLG)-DOX-Ab Gly-Phe-Leu-Gly 270,000 3.3 (wt%) Preclinical ND (59,79–81)
P-(GFLG-DOX)-GalN Gly-Phe-Leu-Gly 25,000/46,000 5.6/1.5 (wt%) Preclinical ND (59,82,90)
P-(GFLG-DOX)-Lac Gly-Phe-Leu-Gly 20,000–32,000 1.4 mol% Preclinical ND (90)
P-(GFLG-DOX)-TriGal Gly-Phe-Leu-Gly 20,000–32,000 2.1 mol% Preclinical ND (90)
Ma-GFLG-DOX Gly-Phe-Leu-Gly NA NA Preclinical ND (91,92)
D2-GFLG-P(DOXH) Gly-Phe-Leu-Gly 215,000 9.2 (wt%) Preclinical ND (91,93)
HMW1D Gly-Phe-Leu-Gly 115,000 7.4 (wt%) Preclinical ND (93)
TET1D Gly-Phe-Leu-Gly 19,600 10.5 (wt%) Preclinical ND (93)
EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOX Ala-Leu-Ala-Leu NA NA Preclinical ND (94–96)
Ac-Phe-Lys-PABC-DOX Phe-Lys 1045.5 52.0 (wt%) Preclinical ND (12)
EMC-Phe-Lys-PABC-DOX Phe-Lys 1133 50.0 (wt%) Preclinical ND (2,18,104)
PG-Phe-Lys-DOX Phe-Lys 1207.8 45.0 (wt%) Preclinical ND (18,41,105)
Z-Phe-Lys-PABC-DOX Phe-Lys 1074.0 50.6 (wt%) Preclinical ND (104)
BR96-SC-Phe-Lys-PABC-DOX Phe-Lys NA NA Preclinical ND (104)

Mw, molecular weight; DOX, doxorubicin; MTD, maximum tolerated dose; NA, not available; ND, not done.