Antibody-directed enzyme prodrug therapy: A review

Many of the limitations of conventional cytotoxic chemotherapy and of attempts to achieve selectivity using antibody vectors can be overcome by a 2 or 3 stage prodrug system. An antibody vectors to tumour sites an enzyme that is not normally present in human extracellular fluids. The tumour located enzyme activates a subsequently administered prodrug. As with other antibody based systems the pharmacokinetics and biodistribution of the antibody-enzyme conjugate are critical elements. In contrast to antibody drug conjugates and radiolabelled antibodies, an enzyme can be inactivated in non-tumour tissues, or subjected to rapid clearance, without toxic effects, which allows high tumour to normal tissue ratios to be achieved. Enzymes conjugated to antibodies increase the problem of immunogenicity and require either the use of immunosuppressive agents, or the development of non-immunogenic catalysts. A small scale pilot clinical trial has shown the general feasibility of the approach. © 1995 Wiley-Liss, Inc.     

Viral-based gene delivery and regulated gene expression for targeted cancer therapy

Importance of the field: Cancer is both a major health concern and a care-cost issue in the US and the rest of the world. It is estimated that there will be a total of 1,479,350 new cancer cases and 562,340 cancer deaths in 2009 within the US alone. One of the major obstacles in cancer therapy is the ability to target specifically cancer cells. Most existing chemotherapies and other routine therapies (such as radiation therapy and hormonal manipulation) use indiscriminate approaches in which both cancer cells and non-cancerous surrounding cells are treated equally by the toxic treatment. As a result, either the cancer cell escapes the toxic dosage necessary for cell death and consequently resumes replication, or an adequate lethal dose that kills the cancer cell also causes the cancer patient to perish. Owing to this dilemma, cancer- or organ/tissue-specific targeting is greatly desired for effective cancer treatment and the reduction of side effect cytotoxicity within the patient.

Areas covered in this review: In this review, the strategies of targeted cancer therapy are discussed, with an emphasis on viral-based gene delivery and regulated gene expression.

What the reader will gain: Numerous approaches and updates in this field are presented for several common cancer types.

Take home message: A summary of existing challenges and future directions is also included.     

Novel gene-directed enzyme prodrug therapies against prostate cancer

There is no effective cure for late-stage hormone (androgen) refractory prostate cancer. Although chemotherapy offers palliation to these late-stage patients, it also leads to systemic toxicities leading to poor quality of life. Clearly, the focus is on the development and evaluation of novel biologically relevant alternatives such as cytoreductive gene-directed enzyme prodrug therapy (GDEPT). With the current limitations of effective gene delivery in vivo, the in situ amplification of cytotoxicity due to bystander effects of GDEPT has special attraction for patients with prostate cancer, the prostate being dispensable. This review focuses on the development, application and potential of various GDEPTs for treating prostate cancer. The current status of research related to the issues of enhancement of in situ GDEPT delivery and prostate cancer-specific targeting of vectors (especially viral vectors) is assessed. Finally, the scope and progress of synergies between GDEPT and other treatment modalities, both traditional and alternate, are discussed.     

Enzyme prodrug therapy of cancer

Tumour therapy may be greatly improved if an enzyme is used to activate a relatively non-toxic prodrug into a potent cytotoxic drug locally at the tumour site, since this should result in increased drug delivery to the tumour and reduced drug levels in critical normal tissues. Antibody-directed enzyme prodrug therapy (ADEPT) utilises the specificity of tumour selective monoclonal antibodies to achieve selective enzyme delivery to the tumour. Viral-directed enzyme prodrug therapy (VDEPT) and gene-directed enzyme prodrug therapy (GDEPT) utilise promoter regulated enzyme gene expression to achieve similar selectivity. Both approaches have been shown to be capable of achieving major improvements in tumour therapy in animal tumour models when compared with conventional systemic delivery of cytotoxic drugs. Early clinical trials with first generation ADEPT and VDEPT/GDEPT systems suggest that the effects achieved in animal tumours will translate well into the clinic. This article describes the development of these approaches over the last 10 years, reviewing both primary and patent literature.     

Enzymatic stability of 2'-ethylcarbonate-linked paclitaxel in serum and conversion to paclitaxel by rabbit liver carboxylesterase for use in prodrug/enzyme therapy

In prodrug/enzyme therapy for cancer, information on the sensitivity of hydrolytic enzymes to prodrug is required to reduce adverse effects of the parental drug and to find the activating enzyme. The aim of this study was to characterize the enzymatic stability of 2'-ethylcarbonate-linked paclitaxel (TAX-2'-Et) in the sera of several different species including humans. TAX-2'-Et disposition in serum was kinetically analysed using models with hydrolytic and/or degradation processes. To further evaluate the capability of liver carboxylesterases (CESs) in TAX-2'-Et hydrolysis, a CES isolated from rabbit liver (Ra-CES) was utilized as a model enzyme. Rat serum provided rapid enzymatic hydrolysis of TAX-2'-Et with a half-life of 4 min. The degradation of paclitaxel (TAX) (degradation rate constant, 0.16 h(-1)) was accompanied by the formation of an unknown compound. The conversion to TAX was almost completely inhibited by phenylmethyl sulfonylfluoride (PMSF) and bis(p-nitrophenyl) phosphate (BNPP). In human and rabbit sera, the degradation rate constant of TAX-2'-Et was 5.1 x 10(-2) and 0.15 h(-1), respectively, when excepting hydrolysis. The degradation products had the same molecular weight as TAX-2'-Et. The amount of TAX produced accounted for only 8-11% of the decrease in TAX-2'-Et after a 9 h exposure to rabbit or human serum. PMSF, but not BNPP, inhibited more than 90% of the TAX production in a 1.5 h incubation with human or rabbit serum. Ra-CES enzyme converted TAX-2'-Et to TAX with V(max) and K(m) of 74.7+/-13.8 nmol/min/mg protein and 8.8+/-2.8 microM, respectively. These results indicate that TAX-2'-Et is sensitive to serum CESs, but not cholinesterases. However, serum CESs show species-dependent hydrolysis of TAX-2'-Et. Although human serum allows the slow release of TAX, TAX-2'-Et is expected to reduce the side-effects of TAX. The Ra-CES enzyme is capable of hydrolysing TAX-2'-Et, which may be beneficial for the development of a TAX-2'-Et/enzyme therapy strategy for ovarian cancer.     

Lectin-directed enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs

The lectin-directed enzyme activated prodrug therapy (LEAPT) bipartite drug delivery system utilizes glycosylated enzyme, localized according to its sugar pattern, and capped prodrugs released by that enzyme. In this way, the sugar coat of a synthetic enzyme determines the site of release of a given drug. Here, prodrugs of doxorubicin and 5-fluorouracil capped by the nonmammalian l-rhamnosyl sugar unit have been efficiently synthesized and evaluated for use in the LEAPT system. Both are stable in blood, released by synthetically d-galactosylated rhamnosidase enzyme, and do not inhibit the uptake of the synthetic enzyme to its liver target. These results are consistent with their proposed mode of action and efficacy in models of liver cancer, and confirm modular flexibility in the drugs that may be used in LEAPT.     

抗肿瘤前药在肿瘤靶向治疗中的新进展

化疗是临床上治疗肿瘤的主要途径之一,但化疗药物在肿瘤部位的低浓度及系统毒性仍是其临床应用受限的首要问题。前体药物通过对原药的化学结构或药物剂型给予一定修饰,大大克服了化疗药物的缺点,开辟了一条靶向治疗肿瘤的新途径。目前,新发展的抗肿瘤前药有导向酶活化前药、载体前药、脂质前药等,现对其研究进展和应用做一综述。     

Paclitaxel-2′-Ethylcarbonate Prodrug Can Circumvent P-glycoprotein-mediated Cellular Efflux to Increase Drug Cytotoxicity

Purpose The aim of the study was to investigate whether 2′-ethylcarbonate-linked paclitaxel (TAX-2′-Et) circumvents P-glycoprotein (P-gp)-mediated cellular efflux and cytotoxicity enhanced by TAX-2′-Et activation within human culture cells transfected with a rabbit liver carboxylesterase (Ra-CES) cDNA. Materials and Methods TAX-2′-Et transport was characterized in a human colon carcinoma cell line (Caco-2) and paclitaxel (TAX)-resistant ovarian carcinoma cells (SKOV3/TAX60). Expression of P-gp, multidrug resistance protein (MRP) 2 and Ra-CES was detected by Western blotting. Cytotoxicity against Ra-CES-expressing cells and cellular amount of TAX produced were determined by MTT assay and using HPLC, respectively. Results Unlike rhodamine123 and TAX, TAX-2′-Et did not exhibit polarized transport in the Caco-2 cells in the absence or presence of verapamil. P-gp levels were expressed much higher in the SKOV3/TAX60 cells than in the Caco-2 cells. MRP2 protein was not detectable in the SKOV3/TAX60 cells. Uptake by the SKOV3/TAX60 cells was similar in quantity to the amount internalized by P-gp-negative SKOV3 cells. In the SKOV3/TAX60 cells, cellular uptake of TAX-2′-Et was not altered regardless of the absence or presence of verapamil. The cytotoxicity to the untransfected SKOV3 cells induced by TAX-2′-Et was significantly lower than that induced by TAX. In the Ra-CES-expressing SKOV3 line, the EC₅₀ value of TAX (10.6 nM) was approximately four-fold higher than that of TAX-2′-Et (2.5 nM). Transfection of Ra-CES into another TAX-resistant ovarian carcinoma cells (KOC-7c) conferred a high level of TAX-2′-Et cytotoxicity via prodrug activation. The intracellular levels of TAX produced from TAX-2′-Et in the Ra-CES-positive KOC-7c cells significantly increased compared with the levels seen in exposure of the untransfected KOC-7c cells to TAX. Conclusions TAX-2′-Et can circumvent P-gp-associated cellular efflux of TAX. TAX-2′-Et is converted into TAX by the Ra-CES, supporting its potential use as a theoretical GDEPT strategy for cancer cells expressing high levels of P-gp. The TAX-2′-Et prodrug efficiently increased the amount of intracellular TAX, which mediates tumor cell death.     

Antibody-directed enzyme prodrug therapy (ADEPT): a review

Antibody-directed enzyme prodrug therapy (ADEPT) is a therapeutic strategy which aims to improve the selectivity of anticancer drugs. ADEPT is a two-step antibody targeting system that has benefits over a one-step chemo-, toxin- or radioimmunoconjugate. The basic principles of ADEPT are discussed alongside the requirements of the components: antibodies, enzymes and prodrugs. The design and syntheses of prodrugs are detailed particularly prodrug/drug systems of potential clinical use, the rationale behind their design and the in vitro and in vivo results obtained. The main features of ADEPT, such as targeting of cancer cells by the antibody-enzyme conjugates, enzymic activation of the prodrugs, selection of the prodrug/drug and enzyme/prodrug systems are reviewed.     

硝基苯并环磷酰胺化合物的还原及其细胞增殖抑制活性的研究

目的对以基因导向酶前药治疗策略为目的所合成的硝基环磷酰胺类化合物还原性及细胞增殖抑制作用进行研究。方法以NaBH4化学还原和E .coli硝基还原酶进行还原性研究;以E .coli硝基还原酶(T116 )和人醌氧化还原酶(NQO1F179)表达细胞(V79)进行细胞增殖抑制作用研究。结果以氧原子与硝基苯环相连的环磷酰6b和6d对E .coli硝基还原酶表达细胞增殖毒性的选择性在30倍以上。结论6b和6d为进一步通过结构修饰筛选选择性毒性的基因导向性肿瘤治疗药物提供了一类新的结构类型     

Nitroreductase: a prodrug-activating enzyme for cancer gene therapy

1. The prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) is activated by Escherichia coli nitroreductase (NTR) to a potent DNA-crosslinking agent. 2. Virus-mediated expression of NTR in tumour cells sensitizes them to CB1954 in vitro and in vivo, providing the basis for a strategy of cancer gene therapy. 3. A phase I trial of CB1954 in cancer patients has been completed, documenting the pharmacokinetics and establishing an acceptable dose. Subsequent trials of the replication-defective adenovirus CTL102 in patients with resectable tumours have documented expression of NTR in injected colorectal liver metastases, hepatocellular carcinoma, head and neck cancer and prostate cancer. Trials combining CTL102 and CB1954 are underway. 4. An oncolytic (replication-competent) adenovirus vector allowed increased expression of NTR in vitro and in a mouse tumour model, resulting in a greater reduction in tumour growth when combined with CB1954 treatment. 5. Alternative prodrugs may eventually prove superior to CB1954; a nitroaryl phosphoramide mustard prodrug activated by NTR shows a greater therapeutic index than CB1954 in a human ovarian carcinoma. 6. The crystal structure of NTR provided the basis for site-directed mutagenesis, which has identified a number of mutants with improved kinetics of CB1954 activation. These can provide improved cell sensitization to CB1954. Combinations of these are being tested. 7. The basis for a positive selection for improved NTR variants has been demonstrated.     

丹参注射液对自杀基因tk/GCV系统的协同增效作用

目的探讨丹参注射液联合自杀基因系统对大鼠肝癌细胞和小鼠移植瘤的作用。方法①丹参注射液以不同浓度与GCV分别或共同作用于大鼠肝癌CBRH7919的tk-细胞,以及含5%tk+的tk+、tk-混合细胞,MTT法检测各组存活率,用Q值(实测药效与理论药效的比值)分析中药与自杀基因系统联合的相互作用是否有协同性,0.85≤Q<1.15为相加;Q≥1.15为协同。②把小鼠肝癌细胞H22/tk(tk+)与H22(tk-)细胞按1∶4混合,再按2×106细胞/只接种到Balb/c纯系小鼠腋下皮下组织内,随机分为模型组、丹参(注射液)组、tk/GCV组、tk/GCV联合丹参注射液治疗的联合组,n=9~11;中药治疗从d 2起共15 d,自杀基因系统治疗从长出肿瘤后开始共10 d,进行疗效观察。结果①各组存活率tk/GCV组为63.10%±2.17%,GCV组为67.03%±2.81%,5 ml.L-1丹参联合tk/GCV组为26.67%±4.23%(Q=1.22),5 ml.L-1丹参+GCV组为42.17%±5.36%(Q=1.04);10 ml.L-1丹参联合tk/GCV组为18.10%±5.56%(Q=1.26),10 ml.L-1丹参+GCV组为33.84%±9.84%(Q=1.06)。②接种后各组均在d 7触摸到肿瘤,成瘤率100%。联合组在治疗后肿瘤生长呈现明显抑制,最终肿瘤体积(1.53±0.88)cm3,比模型组(3.19±1.76)cm3减少52.01%(P<0.05);肿瘤质量(1.32±0.80)g,比模型组(2.28±1.20)g减少42.11%(P<0.05),差异有统计学意义。丹参组、tk/GCV组肿瘤体积、瘤块质量与模型组比较差异均无统计学意义。结论丹参注射液能提高自杀基因系统对大鼠肝癌细胞的杀伤力和对小鼠肝癌细胞移植瘤的抑制作用,两者联合作用的协同性提示丹参注射液能增强自杀基因旁观者效应。     

胞嘧啶脱氨酶基因与前体药物5-氟胞嘧啶对人肝癌的基因治疗作用(英文)

目的:探讨胞嘧啶脱氨酶基因(CD)与前体药物5-氟胞嘧啶(Flu)对肝癌的抗肿瘤作用,方法:应用逆转录病毒法,将CD基因转导入肿瘤细胞,体外应用克隆分析法测定Flu对肿瘤细胞的细胞毒性;应用裸鼠移植瘤模型研究CD基因/Flu体系对肝癌的抗肿瘤作用,结果:前体药物Flu对人肝癌在体外和裸鼠体内具有明显的抗癌作用.与5-氟尿嘧啶10 mg·kg~(-1)相比,前体药物Flu 500 mg·kg~(-1)对裸鼠肝癌移植瘤有更强的抗癌作用.CD基因/Flu对人肝癌裸鼠移植瘤具有旁观者杀伤效应.结论:CD基因/Flu体系在治疗肝癌中有潜在的价值。     

Antibody‐Directed Enzyme Prodrug Therapy: A Promising Approach for a Selective Treatment of Cancer Based on Prodrugs and Monoclonal Antibodies

The antibody‐directed enzyme prodrug therapy allows a selective liberation of cytotoxic agents from non‐toxic prodrugs in cancerous tissue by targeted antibody–enzyme conjugates. We have developed a series of novel glycosidic prodrugs based on the natural antibiotic CC‐1065 and the duocarmycins, which are up to 4800 times less toxic than the drugs liberated from these prodrugs in the presence of the activating enzyme (e.g., β‐d ‐galactosidase). Furthermore, the drugs show very high cytotoxicities with IC₅₀ values of as low as 4.5 pm . In this report, we summarize our recent results on the development and biological evaluation of these novel third‐generation prodrugs with higher water solubility, higher difference in cytotoxicity between the prodrugs and the corresponding drugs and improved cytotoxicity of the drugs as compared with previous compounds.     

Patent property of suicide gene therapy involving prodrugs 1996 - 1999

Suicide gene therapy can be defined as a two-step treatment for solid tumours. In the first step, specific gene delivery technology leads to the expression of a foreign prodrug-activating enzyme only in tumour cells. During the second step, a non-toxic prodrug is administered which is converted by the expressed enzyme to a potent cytotoxic agent. The patent literature covering new gene-directed enzyme prodrug therapy (GDEPT) systems, improvements to GDEPT systems and improved delivery for suicide gene therapy, is discussed.     

GSH-sensitive polymeric prodrug: Synthesis and loading with photosensitizers as nanoscale chemo-photodynamic anti-cancer nanomedicine

Precisely delivering combinational therapeutic agents has become a crucial challenge for anti-tumor treatment. In this study, a novel redox-responsive polymeric prodrug (molecular weight, MW: 93.5 kDa) was produced by reversible addition–fragmentation chain transfer (RAFT) polymerization. The amphiphilic block polymer-doxorubicin (DOX) prodrug was employed to deliver a hydrophobic photosensitizer (PS), chlorin e6 (Ce6), and the as-prepared nanoscale system [NPs(Ce6)] was investigated as a chemo-photodynamic anti-cancer agent. The glutathione (GSH)-cleavable disulfide bond was inserted into the backbone of the polymer for biodegradation inside tumor cells, and DOX conjugated onto the polymer with a disulfide bond was successfully released intracellularly. NPs(Ce6) released DOX and Ce6 with their original molecular structures and degraded into segments with low MWs of 41.2 kDa in the presence of GSH. NPs(Ce6) showed a chemo-photodynamic therapeutic effect to kill 4T1 murine breast cancer cells, which was confirmed from a collapsed cell morphology, a lifted level in the intracellular reactive oxygen species, a reduced viability and induced apoptosis. Moreover, ex vivo fluorescence images indicated that NPs(Ce6) retained in the tumor, and exhibited a remarkable in vivo anticancer efficacy. The combinational therapy showed a significantly increased tumor growth inhibition (TGI, 58.53%). Therefore, the redox-responsive, amphiphilic block polymeric prodrug could have a great potential as a chemo-photodynamic anti-cancer agent.     

N-乙酰基-L-谷氨酰基-泼尼松龙肾靶向前体药物研究

目的通过研究N-乙酰基-L-谷氨酰基-泼尼松龙的体内分布，考察该前体药物的肾靶向性。方法小鼠iv后，采用高效液相法，在规定时间段测定各组织脏器的泼尼松龙浓度, 并采用大鼠骨密度的测定仪确证前体药物的副作用。结果小鼠给药后15 min，前体药物组肾脏中泼尼松龙浓度为(86±8) μg·g^-1，泼尼松龙组为(57±4) μg·g^-1，60 min后前体药物组肾脏药物浓度为 (67±5) μg·g^-1；泼尼松龙组(42±4) μg·g^-1。大鼠给药30 d后，股骨的骨密度分别为(0.08±0.03) g·cm^-2 （泼尼松龙）和(0.14±0.06) g·cm^-2(前体药物组)。结论前体药物具有肾靶向性, 并能降低致骨质疏松的副作用。     

Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: Effects of combining beneficial single mutations

Prodrug activation gene therapy for cancer involves expressing prodrug-activating enzymes in tumour cells, so they can be selectively killed by systemically administered prodrug. For example, Escherichia colinfsB nitroreductase (E.C. 1.6.99.7)(NTR), sensitises cells to the prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide), which it converts to a potent DNA-crosslinking agent. However, low catalytic efficiency with this non-natural substrate appears to limit the efficacy of this enzyme prodrug combination for eliminating the target cancer cells. To improve this, we aim to engineer NTR for improved prodrug activation. Previously, a number of single amino acid substitutions at six positions around the active site of the enzyme were found to increase activity, resulting in up to ∼5-fold enhanced cell sensitisation to CB1954. In this study we have made pairwise combinations among some of the best mutants at each of these 6 sites. A total of 53 double mutants were initially screened in E. coli, then the 7 most promising were inserted into an adenovirus vector and compared in SKOV3 human ovarian carcinoma cells for sensitisation to CB1954 and two alternative prodrugs. The most effective mutants, T41L/N71S and T41L/F70A, were 14-17-fold more potent than WT NTR at sensitising the cancer cells to CB1954. The best mutant for activation of the dinitrobenzamide mustard prodrug SN23862 was T41L/F70A (4.8-fold improvement); and S40A/F124M showed 1.7-fold improvement over WT with the nitrobenzylphosphoramide mustard prodrug LH7. In two tumour xenograft models using SKOV3 or human prostate carcinoma PC3, T41L/N71S NTR demonstrated greater CB1954-dependent anti-tumour activity than WT NTR.