Other Methods of Directed Enzyme Prodrug Activation

Compared to the above discussed strategies in the field of prodrug therapy the number of publications dealing with LEAPT, PDEPT, MDEPT, and some other approaches, including targeting transporters in prodrug design (Dahan et al., 2014) are rather low; for reviews, see de Albuquerque et al. (2005), Stanczak and Ferra (2006), or Bader et al. (2014).

Lectin-directed enzyme activated prodrug therapy

Lectins are proteins that contain defined carbohydrate recognition domains (CRDs). Via theses CRDs they bind carbohydrates to form carbohydrate-protein complexes. They are among others involved in cell development and signaling or the immune system, as well as states of diseases and malignancies (see Lindhorst, 2016, for a review). LEAPT makes use of highly specific and potent binding mechanisms that are provided by Nature; the approach comprises a prodrug and a lectin-directed glycoconjugate; the glycoconjugate cleaves the prodrug and thereby releases the drug regio-selectively, based on a carbohydrate-lectin interaction-mediated localization of a glycosidase enzyme of the target cells (Zhang and Wang, 2015). An example has been published by Robinson et al. (2004). A a- L-rhamnosidase, an enzyme belonging to the group of glycosyl hydrolases, was engineered through enzymatic deglycosylation and chemical reglycosylation to render it possible for targeting hepatocytes by interaction with the asialoglycoprotein receptor that is expressed in several thousand copies on the liver cell hepatocyte surface. After complete uptake of the engineered rhamnosidase, a prodrug consisting of a drug molecule—in this case, doxorubicin—capped with an L-rhamnoside (Rha) is administered. As the L-rhamnopyranose (naringinase, N) is of non-mammalian origin (De Liese et al., 2016), the Rha-capped prodrugs cannot be processed by mammalian enzymes which means that doxorubicin is released primarily by the prelocalized a-L-rhamnosidase. The in vivo therapeutic efficacy was confirmed by a near halving in total tumor burden in nude athymic mice that received an injection of the human hepatocellular liver carcinoma cell line HepG2 prior to treatment with the drug/ sugar conjugate. An advantage of LEAPT is that by altering the glycosylation pattern the enzyme may be easily redirected to other cell types. For the application of 5-fluorouracil capped by the non-mammalian L-rhamnosyl sugar and the release of 5-fluorouracil by the action of synthetically D-galactosylated rhamnosidase enzyme together with investigations into the efficacy of this approach in models of liver cancer, see Gamier et al. (2010).

Polymer-directed enzyme prodrug therapy and polymer enzyme liposome therapy

In these prodrug therapies, the prodrug is targeted to tumors by passive processes. The drug is linked to inert macromolecular carriers as, e.g., synthetic or biopolymers or nanoparticles such as liposomes or nanospheres acting as inert carriers that must be non-toxic and non-immunogenic. Drug-conjugation to hydrophilic polymeric carriers improves the water solubility of hydrophobic drugs such as doxorubicin and paclitaxel with advantages for formulation and patient administration. These nanoparticulated drugs exploit the leaky or defective neovasculature in tumors being significantly different from the respective architecture of normal blood cells and characterized by large pore sizes (> 100 nm) of tumor microvessels that enable the uptake of polymers the radii of which are normally below 10 nm, whereas their extravasation in normal cells is not possible (Singh et al., 2008). Drug conjugates developed in this connection are termed nanomedicines. According to European Science Foundation's Forward Look on Nanomedicine they comprise "nanometer size scale complex systems, consisting of at least two components, one of which being the active ingredient” (European Science Foundation, 2005). Macromolecular polymer therapeutics are not bioavailable orally; polymer-protein conjugates are administered subcutaneously or intramuscularly, in contrast to polymer-drug conjugates given intravenously. In some cases such drugs are given locally, e.g., through the hepatic artery to patients with liver cancer.

Polymer-directed enzyme prodrug therapy

Polymer-drug conjugates are applied as antiviral agents, immunomodulators and for enzyme replacement; this Section focuses on their use in cancer treatment. Polymers that have been applied clinically, are mainly of linear structure such as the below mentioned /V-(2-hydroxypropyl)metha cry 1-amide (HPMA) copolymers, polyglutamic acid (PGA), polysaccharides like dextran and polyethyleneglycol (PEG). Many of these polymer-drug conjugates are used in PEGylated form resulting in increased plasma residence, reduced immunogenicity and increased therapeutic index tantamount to reduced drug toxicity.

Bae et al. (2012) reported the development of a nanogel of excellent biocompatibility as controlled anticancer drug delivery carrier that is based on poly(y-glutamic acid) (y-PGA) produced by microbial species. y-PGA was thiolated by covalent coupling between the carboxyl groups of y-PGA and the primary amine group of cysteamine. After loading with doxorubicin PEG was added resulting in hydrogen-bond interactions between thiol groups of thiolated gamma-PGA and hydroxyl groups of PEG. Ultrasonication led to the formation of disulfide linkages. The average size of DOX-loaded y-PGA in aqueous solution was 136.3 +/- 37.6 nm and the loading amount about 38.7 pg per mg of y-PGA. Controlled drug release was tested in presence of reducing agents such as the tripeptide glutathione (GSH). The authors could show that Dox-loaded у PGA nanogels were distributed in the cytoplasm and nuclei of MCF-7 cells where DOX was released in a controlled manner due to collapsing disulfide cross-linkages under the reducing condition of the cells. Bisht and Maitra (2009) described the potential of dextran-doxorubicin/ chitosan nanoparticles for solid tumor therapy by selectively providing therapeutically effective drug concentrations at the tumor site through enhanced permeability and retention (EPR). They discussed targeted delivery of anti cancer drugs such as doxorubicin, chemically conjugated with dextran and encapsulated in chitosan nanoparticles, to solid tumors.

PEGylated enzymes have found wider applications in cancer therapy with the aim to deprive malignant cells of nutrients such as amino acids essential for their growth, e.g., in connection with protein biosynthesis (see also Chapter 8, Vol. 4 of this Series on Biocatalysis); such a strategy makes use of enzymes that degrade these amino acids as well as of the fact that many cancer

types are deficient in enzymes required for amino acid synthesis; such an enzyme is argininosuccinate synthetase-1 that in a sequential action with argininosuccinate lyase catalyzes the synthesis of arginine from citrulline. PEG-L-asparaginase (Oncaspar) was the first to achieve US FDA approval in 1994 for the treatment of acute lymphoblastic leukemia and lymphoma. L-asparaginase depletes the amino acid asparagine (see the scheme on the previous page) that is essential for tumor growth. PEG-recombinant arginine deiminase (PEG-rhADI) has also been reported to inhibit in vivo and in vitro proliferation of hepatocellular carcinoma cells (Cheng et al., 2007) because this enzyme catabolizes arginine to citrulline (see the scheme on the previous page). Miraki-Moud et al. (2015) found that arginine deprivation using PEGylated arginine deiminase (ADI-PEG 20) has activity against primary acute myeloid leukemia cells in vivo. The application of ADI-PEG 20 in combination with cytarabine (cytosine arabinoside, a chemotherapy medication) was more effective than either treatment alone. A review published by Feun et al. (2015) deals with recent studies that focus on Arg-dependent malignancies with arginine-degrading enzymes, including arginase and arginine deiminase as well as a discussion of mechanisms of resistance that cancers develop after such drug exposure. The potential of arginine deiminase as an antitumor agent has been reviewed by Somani and Chaskar (2018). There is an increasing interest in the development of novel types of human arginase for targeting arginine-dependent cancers, in particular a pegylated enzyme (hArg I [Co]-PEG5000) that has shown good efficacy in depleting arginine and where the two essential manganese (II) ions are replaced with cobalt (Phillips et al., 2013). Khoury et al. (2015) presented a study according to which human recombinant arginase I (Co)-PEG50oo is selectively cytotoxic to human glioblastoma cells. For an early review that in addition focuses on the mechanism of action of drug conjugates, see Duncan (2006). A more recent review is from Scomparin et al. (2017) and discusses advances and future perspectives of both polymer-directed enzyme prodrug therapy (PDEPT) and polymer enzyme liposome therapy (PELT); the latter is briefly treated in the next Section.

Polymer enzyme liposome therapy

Liposomes are comprised of an aqueous solution core that is surrounded by a hydrophobic lipid bilayer membrane made up by phospholipids such as phosphatidylcholine, hence enabling a loading of liposomes with hydrophilic and/or hydrophobic molecules/drugs. The lipid bilayer of a liposome can fuse with cell membranes a process used to deliver drugs that cannot pass cell membranes by diffusion, inside of cells. For reviews of clinical developments of liposome-based drugs/formulations, see Chang and Yeh (2012) and Bulbake et al. (2017). Polymer enzyme liposome therapy is a theoretical option for treating diseases such as cancer and as the other strategies presented in this chapter represents a two-step approach, too. First investigations with a liposomal drug and a polymer-phospholipase conjugate were performed by Ferguson et al. (2017) with a liposomal drug and a polymer-phospholipase (phospholipases C (PLC) and PLA2) conjugate administered sequentially in connection with a potential cancer treatment. The two PL-conjugates were tested with respect to their ability to trigger the release of anticancer anthracyclines doxorubicin from polyethyleneglycol-coated (PEGylated) Caelyx® and daunomycin from non-PEGylated DaunoXome®. Caelyx® has been approved by the EMA and the FDA for treatment of recurrent/Pt-resistant ovarian cancer. PEGylation increases the plasma half-life and the liposomes can extravasate through the leaky tumor vasculature; in contrast to free doxorubicin, cardiotoxicity has been shown to be extremely rare allowing higher doses of the drug (Green and Rose, 2006; Pisano et al., 2013). DaunoXome is approved for the treatment of patients with advanced HIV-related Kaposi's Sarcoma. Ferguson et al. performed their experiments with PLC conjugated with N- (2-hydroxypropyl)methacrylamide (HPMA) as well as with a PLA2-dextrin conjugate. HPMA was developed by Vasey et al. (1999) as the first member of this class of chemotherapeutic agents-drug-polymer conjugates. The conjugate comprises doxorubicin covalently bound to the copolymer by a peptidyl linker; after cellular uptake the linker is cleaved by lysosomal enzymes, resulting in intratumoral drug release. Ferguson et al.

found among others that both enzyme polymer preparations released daunomycin rather rapidly, whereas doxorubicin release from Caelyx® was relatively slow. Furthermore, the dextrin-PLA2 conjugate potentiated the cytotoxicity of DaunoXome® to MCF-7 (a breast cancer cell line) to a greater extent than free PLA2.

Melanocyte-directed enzyme prodrug therapy (MDEPT)

According to Siegel et al. (2018) the estimated number of new cases of malignant melanoma, a highly aggressive form of skin cancer, is 91,270 (55,150 males and 36,120 females) in the USA alone, with a death rate of 10%. Lowe et al. (2014) found that the incidence of cutaneous melanoma among middle-aged adults in the USA increased over the past 4 decades, which is in agreement with reports from other countries, but that disease-specific mortality has decreased, particularly in the last 10 years probably due among others to earlier detection through educational programs, and more skin cancer screenings. Melanoma is a complex and genomically diverse malignancy that has been reviewed by Lin and Fisher (2017) with respect to understanding of signaling and immune regulation in melanoma and implications for responses to treatment by multiple targeted therapies and immunotherapies that have meanwhile entered the clinic. This kind of cancer that in rare cases also occur in the mouth,

intestines, or eye, develops from the melanin-containing so-called melanocytes; the skin pigment protects from UVB radiation (Riley, 2003). Among the different types of melanin the most common one is eumelanin with part of its structure shown in the scheme on the previous page; structural building blocks are among others 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and L-dopamine; the arrows indicate possible sites of polymer growth. According to the Raper-Mason pathway (Mason, 1948) eumelanin formation results from the tyrosine hydroxylase and dopa oxidase activity of tyrosinase, an enzyme containing a pair of antiferromagnetically coupled copper ions in its active site. It is present in plant and animal tissues as well as in bacteria (e.g., Streptomyces sp.). The two Cu ions surrounded by six His residues bind one molecule of atmospheric oxygen, required for catalysis: the intermediate L-dopaquinone is formed during the reaction cycles to L-leukodopachrome and reacts with L-dopaquinone to L-dopachrome which is converted to the two eumelanin structural units DHI and DHICA. Further enzyme-catalyzed oxidation of these dihydroxyindoles gives indolequinones; polymer formation occurs via cross-linking reactions (Solano, 2014). A review of structure-function correlations in tyrosinases belonging to the type-3 copper protein family has been provided by Kanteev et al. (2015).

Among the drugs used today for chemotherapeutic melanoma treatment are temozolomide carboplatin and paclitaxel or immunotherapeutic drugs such as ipilimumab, pembrolizumab, vemurafenib, trametinib, and peginterferon alfa-2b (e.g., Peglntron) (Batus et al., 2013) which is also used for treating Hepatitis C. In its PEGylated form it has been approved by the US FDA and is on the World Health Organization's List of Essential Medicines. The lipophilic Temozolomide represents a prodrug that is stable at acidic pH values so that it can be administered orally. After absorption it rapidly undergoes spontaneous formation

of monomethyl triazene 5-(3-methyltriazen-l-yl)-imidazole- 4-carboxamide as an intermediate that reacts with water to liberate 5-aminoimidazole-4-carboxamide and the highly reactive methyldiazonium cation. The latter acts by methylating DNA mainly at N-7 positions of guanine in guanine rich regions but also at N-3 adenine, and 0-6 guanine residues (for more information, see Zhang et al., 2012, and literature cited therein).

Another melanoma treatment strategy focuses on pro drugs that are activated by the tyrosinase enzyme which is upregulated within malignant melanomas/mutant melanocytes compared with healthy melanocytes, hence providing an in-situ tool for the activation of melanoma prodrugs (Jawaid et al., 2009). Perry et al. (2009) developed and characterized a range of triazene derivatives as prodrug candidates for melanocyte-directed enzyme prodrug therapy (MDEPT). The prodrugs contained a tyramine or dopamine promoiety required for tyrosinase activation, joined via an urea functional group to the cytotoxic triazene. Recently Sousa et al. (2017) reported the synthesis of new DNA-alkylating triazene derivatives containing hydroxyphenylalkyl carboxylic acids, activated by tyrosinase for application to a melanoma-specific therapy. The investigations included structure-activity relationship studies enabling the identification of optimal structural features for enzyme affinity.

Jordan and co-workers (2001) synthesized a series of pro drugs for MDEPT using as cytotoxic agents phenyl mustard, bisethyl amine mustard and daunomycin compounds with the latter shown in the

scheme on the previous page; in all cases a carbamate, urea or thiourea linker was used between the moiety serving as tyrosinase substrate and the active drug. In a more recent paper Webster et al. (2014) describe a bisphosphonamidate clodronate prodrug that showed efficient cellular uptake and intracellular activation with selective cytotoxic activity in melanoma cells. The action mechanism of bisphosphonates concerning the treatment of malignancies has been reviewed by Van Acker et al. (2016). A review article describing the advances in the development of targeted prodrugs for the treatment of malignant melanoma has been recently published by Machado et al. (2016). Finally Calado et al. (2016) incorporated the triazene prodrug (TPD) 3-[(2-(acetylamino)- 3-(4-hydroxyphenylpropanoyl)]-l-(4-ethoxycarbonylphenyl)-3- methyltriazene that had been demonstrated to be good substrate for tyrosinase in liposomes where it is. The prodrug (scheme above) is composed among others of a transporter unit, the IV-acetyl-L-tyrosine residue, which is a good substrate for the tyrosinase, and of the hydrolysis product monomethyltriazene (MMT) triazene. The liposomal formulations were prepared from different phospholipids (pure ones, egg phosphatidylcholine (PC), phosphatidylglicerol, distearoyl phosphatidylethanolamine, etc.), covalently linked to PEG2000. In vitro tests in a malignant human melanoma cell line revealed that liposomal incorporation of the triazene prodrug into egg-PC potentiated its cytotoxic effect against a human malignant melanoma cell line in a way being superior compared to the clinically used Temozolomide.

 
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