Prodrug Approaches for Improved DMPK Characteristics and Their Preclinical Study Considerations

Prodrugs refer to a class of compounds without or with poor activity, but can generate active metabolites (parent drug) with enhanced pharmacological activity after biotransformation. Prodrug strategy is a common and effective approach in drug screening and lead compound optimization. Generally, the drug structures are modified or covalently linked to carrier molecules to improve the physicochemical characteristics and address issues related to absorption, distribution, metabolism, excretion, and toxicity.

Approximately 10% of drugs available in the global market are classified as prodrugs, and it is anticipated that more prodrugs will be introduced in the future. This article will provide an overview of the application of prodrugs and their activation strategies, focusing on: #1. Classification of prodrugs based on bioactivation; #2. Impacts of prodrug strategies on drug physicochemical and pharmacokinetic properties; #3. Important considerations for preclinical study on prodrugs.

Table 1. Overview of certain prodrugs approved by the FDA ^{[1, 2]}. The carrier groups are circled.

Classification of prodrugs based on bioactivation

In most cases, based on known biotransformation, chemical modification or derivatization of the parent drug to carry out the corresponding prodrug structural design, through enzyme or non-enzyme mediated activation to release the active drug and exert its pharmaceutical effect. According to the difference in structure and activation mechanism, prodrugs can be divided into carrier-linked prodrugs and bioprecursor prodrugs. The schematic diagrams are as follows.

Figure 1. Schematic diagram of carrier-linked prodrugs and bioprecursor prodrugs ^[3]

Carrier-linked prodrugs

The carrier-linked prodrug contains a nontoxic carrier group covalently linked to the parent drug molecule. Common functional groups to be connected in the parent drug are hydroxyl, amino, carboxyl, thiol, and aldehyde groups, thereby forming new functional groups such as ester bonds, carbonates, amides, phosphates, carbamates, etc. (Figure 2).

Figure 2. Functional groups commonly used in the structural design of carrier-linked prodrugs ^[4]

Ester designs are the most commonly employed among carrier-linked prodrugs, accounting for over 50% of marketed prodrugs. Ester bonds can be hydrolyzed by esterases in blood, liver, and other organs or tissues, releasing the active molecules. Ethyl esters, aryl esters, diol esters, cyclic carbonates, and lactones are relatively common ester prodrugs. Telotristat etiprate, a tryptophan hydroxylase inhibitor, is one of the ester prodrugs (Figure 3). After oral administration in humans, the ethyl ester functional group was hydrolyzed by carboxylesterase in vivo and rapidly converted to the telotristat (active drug), while minimal exposure of the telotristat prodrug in the systemic circulation.

Figure 3. Hydrolysis of the ester prodrug ^[5]

The launch of fosphenytoin has drawn extensive attention for phosphoester-type prodrugs. Fosphenytoin is hydrolyzed by alkaline phosphatase in the gastrointestinal tract to produce the hydroxylmethylamine intermediate and inorganic phosphate. This intermediate is unstable and rapidly decomposes into the amine-based drug substance (phenytoin) and an equivalent amount of formaldehyde, which crosses the gastrointestinal mucosa and is absorbed into the systemic circulation (Figure 4). Phosphate prodrugs are more commonly designed for drugs administered parenterally. In addition to gastrointestinal fluid, abundant phosphoesterases in plasma and liver tissue can also release active drugs from phosphate prodrugs.

Figure 4. Phosphate ester prodrug fosphenytoin bioactivation ^[6]

While simple esters are favored for prodrug design, the biotransformation of certain alkyl or aryl esters may not be efficiently catalyzed by esterases, leading to insufficient systemic exposure. By introducing a diester group, the prodrug molecule is more easily recognized and activated through the second esterase site. (Figure 5). After intravenous administration of the diester prodrug isavuconazonium sulfate, it was rapidly and completely hydrolyzed by plasma esterase at the sarcosine ester bond site (red mark). At the same time, the free hydroxyl group attacked another ester bond site in the molecule (blue mark). It underwent intramolecular cyclization to generate the active drug isavuconazole, inactive cyclic by-product (BAL8728), and acetaldehyde. Prodrugs were essentially undetectable in plasma within 0.25 h after 1 h of intravenous infusion, and the exposure to the drug was less than 1% of that of the active drug. The isavuconazole diester prodrug strategy showed an extremely high drug conversion rate.

Figure 5. Hydrolytic activation of diester prodrug isavuconazonium sulfate ^[1]

Compared with ester prodrugs, amides are more stable in enzyme metabolism, which is not conducive to the complete release of active drugs, setting specific limits to their application. However, amide prodrugs can be designed to enhance oral absorption and targeted transport, and hydrolyzed by in vivo proteases or peptidases to release active drugs. The amide prodrug, LY544344 (Figure 6), based on a high affinity of the alanine group to the intestinal epithelial cell oligopeptide transporter PepT1, can be transported into cells, hydrolyzed by peptidase to LY35470, and then passively diffuses into the bloodstream. For amine functional group-based prodrugs, sulfonamides and carbamates are also effective prodrugs that can be hydrolyzed by esterases or amidases.

Figure 6. Hydrolysis of amino acid prodrug LY544344 ^[7]

In addition, some carrier-linked prodrugs are mediated by non-enzyme activation under specific physiological conditions, such as hydrazone prodrugs and N-Mannich base prodrugs. Hydrazone prodrugs are stable in the normal pH range of system circulation while unstable in releasing active drugs in the tissue environment or cell compartment with a lower pH range. The anticancer Aldoxorubicin, after ingestion by tumor cells, releases cytotoxic doxorubicin in the acidic tumor cell compartment (pH 5.5-6.2) and lysosomes (pH 4.5-5.0). Hydrazone bond cleavage in this particular microenvironment makes the release of the active drug more targeted, which can reduce the damage to normal cells caused by cytotoxins. For N-Mannich prodrugs, it decomposes in a neutral and alkaline environment. The release of the active drug will be triggered with increased pH, such as the highly water-soluble Rolitetracycline, which is converted to the active tetracycline at pH 7.4.

Figure 7.pH-dependent carrier-linked prodrugs ^{[2, 6]}

Bioprecursor prodrugs

Unlike carrier-linked prodrugs, bioprecursor prodrugs don’t contain carrier groups, and commonly produce active compounds after metabolism (including oxidation, reduction, phase II conjugation, etc., Figure 8). Bioprecursor prodrugs can be understood as substrates of enzymes and metabolized in vivo to generate expected active products, such as the reversible reduction of the anti-inflammatory drug sulindac to sulfide (Figure 9a) by metabolic enzymes, whose sulfide is the active substance. The antihypertensive drug losartan can also be considered a precursor prodrug and is oxidized to active metabolites in vivo to exert its efficacy (Figure 9b).

Figure 8. Classification of bioprecursor prodrugs based on activation mechanism ^[8]

Figure 9. Metabolic activation of bioprecursor prodrugs: sulindac (a) and losartan (b) ^[3]

Impacts of prodrug strategies on drug physicochemical and pharmacokinetic properties

The design idea of prodrugs is to modify groups or introduce new functional groups based on the parent drug to produce new chemical entities. The generation of such new chemical entities is often accompanied by changes in drug properties, especially when the parent drug has defects in kinetic properties, such as low bioavailability caused by poor lipophilicity or solubility of the drug, insignificant efficacy caused by metabolism, or drug toxicity caused by inaccurate targeting. These problems can be solved by prodrug strategies.

Improving drug solubility

The premise of drug absorption is to require it to be in a dissolved state, and poor water solubility is not conducive to drug absorption and transport in the body. The solubility of drugs can be significantly increased by introducing polar functional groups such as amino acid esters, polyethylene glycol, sugars, or phosphate esters into the drug structure using the prodrug strategy.

Degoey et al. designed the phosphate prodrug of Lopinavir, which has a more than 700-fold increase in aqueous solubility compared with free Lopinavir, and showed that the concentration of active drug released by hydrolysis of the phosphate prodrug was higher in plasma after administration. Lobo et al. synthesized the ester prodrug of diclofenac, which can be rapidly hydrolyzed by esterase to diclofenac in the rat in vitro plasma experiments, and its ester prodrug has better solubility and better transdermal properties than free diclofenac.

Figure 10. Lopinavir phosphate and diclofenac glycerides ^[9]

Effects on drug membrane permeability

The ability of a drug to cross the cell membrane is related to the lipophilicity of the drug and transporter recognition of membrane proteins. To improve drug membrane permeability, common methods are to increase drug lipophilicity through masking polar groups, introducing lipophilic groups, and introducing groups recognizable by transporters.

Oseltamivir, a neuraminidase inhibitor, is the ethyl ester prodrug of RO-64-0802, and the introduction of the ester functional group masks the polar carboxylate group in the molecule. Compared to RO-64-0802, oseltamivir has increased lipophilicity, and its bioavailability has also increased from 5% to 80%. For another neuraminidase inhibitor, zanamivir (Figure 11a), oral bioavailability is very low due to its polar structure, while its amino acid-coupled acyloxy ester prodrug can be recognized by the luminal cell PepT1 transporter, showing better intestinal membrane permeability and bioavailability. The first FDA-approved new coronary pneumonia treatment, remdesivir (Figure 11b), is structurally designed with a phosphoryl amino-ester prodrug. The introduction of aryl esters, alanines, and alkoxy esters allows remdesivir to have good cell membrane penetration, followed by being metabolized intracellularly to tri-phosphorylation product (active drug), and finally interfering with viral RNA transcription.

Figure 11. (a) Transport of the amino acid-conjugated acyloxy ester prodrug of Zanamivir in intestinal epithelial cells ^[8]; (b) intracellular metabolic activation of the phosphoryl amino-ester prodrug remdesivir ^[2]

Improving drug metabolic stability

Metabolic instability is mainly attributed to the presence of readily metabolizable sites in the drug structure that undergo rapid and extensive metabolism in the liver or gastrointestinal tract. This instability reduces the total amount of active drugs entering the systemic circulation and is not conducive to pharmacodynamic exertion. Prodrug strategies can improve drug metabolic stability and protect active drugs from metabolism by modifying the corresponding functional groups to mask easily metabolizable sites.

The bronchodilator bambuterol, a dimethylcarbamate prodrug of terbutaline (Figure 12), has a significantly reduced first-pass metabolic effect due to the protection of the metabolically susceptible phenolic hydroxyl group in the drug structure. Bambuterol is slowly metabolically activated to terbutaline mainly by non-specific butyrylcholinesterase. Due to its slow release and duration of action, bambuterol can achieve once-daily dosing with a lower incidence of adverse effects than terbutaline three times daily dosing.

Figure 12. Metabolic activation of carbamate prodrug bambuterol ^[1]

Improving drug targeting

Whether the drug can be delivered to the target organ is the basis for the drug to exert its efficacy, and targeted modification of the drug structure to achieve selective tissue delivery can improve the drug's therapeutic activity and reduce side effects. The targeting design of prodrugs is often achieved by conjugating the structural units of targeted tissues or cells with cytotoxic molecules. For example, peptide-drug conjugated carrier-linked prodrugs covalently attach peptides to drugs through different linkers to form multifunctional PDCs targeting tumor cells, which can selectively accumulate in tumor cells, have a prolonged half-life, and enhanced efficacy. Antibody-drug conjugated carrier prodrugs utilize the high affinity of monoclonal antibodies for tumor cell surface antigen proteins to target tumor cells to release active drugs through receptor-mediated endocytosis. In addition, the design of anticancer drugs conjugated to active drugs using folic acid or DNA aptamer strands is also a successful case in the targeted application of prodrugs.

Figure 13. Prodrug design with tumor cell-targeted delivery function ^[10]

Special considerations for the preclinical study of prodrugs

The prodrug plays a therapeutic role in the body by being converted into the parent drug (active). As a new chemical entity, the pharmacodynamics and pharmacokinetics of the prodrug cannot be simply speculated by the characteristics of the parent drug. The study of prodrugs requires a full understanding of the physical, chemical, and biological properties of the parent drug. On this basis, the prodrug strategy can be used to optimize the shortcomings of the parent drug without reducing the therapeutic effect. Therefore, it is necessary to comprehensively evaluate prodrug physical and chemical properties, pharmacological effects, kinetic properties, pharmacodynamic effects, and possible toxicities ^[11].

Stability testing of prodrug

For the design of the prodrug, it is necessary to ensure that it can degrade and release the parent drug at the appropriate time and site. This requires that the designed prodrug has appropriate chemical and enzyme stability, which cannot be degraded before reaching the target site, nor be delayed late in degradation after entering the target site, but still exists in the form of a prodrug. Otherwise, it cannot exert its efficacy.

Appropriate in vitro transformation studies can be selected according to the administration route and possible transformation route to investigate the stability of the prodrug:

1. In vitro transformation experiments under different pH conditions, such as artificial intestinal fluid or gastric fluid stability experiments, to determine the transformation rate and degree of prodrugs under various pH conditions.
2. In vitro transformation experiments under different enzyme conditions, such as esterase or peptidase hydrolysis stability experiments, to determine the conversion rate and degree of prodrug hydrolysis into the parent drug.
3. Biotransformation experiments under different biological matrix conditions, such as liver homogenate, plasma, liver microsomes, hepatocytes, intestinal microsomes, and intestinal S9, and other stability experiments, select the appropriate matrix to determine the transformation rate of the prodrug and the possible transformation site.

Comparison with the parent drug

When prodrugs are administered through various routes, it is advisable to concurrently administer the parent drug intravascularly at the whole animal level to determine the absolute bioavailability of the parent drugs in the body after dosing the prodrug. In addition, under the condition of equimolar dose administration as far as possible, the kinetic parameters of the parent drug control group should be provided and compared with the corresponding parameters of the prodrug after administration in vivo to investigate the optimization effect of the prodrug on the pharmacodynamic or pharmacokinetic properties.

Possible toxicity problem of the prodrug

The prodrug is chemically different from the parent drug, which is likely to introduce toxicity. On the one hand, the prodrug may be metabolized before the decomposition and release of the parent drug, forming unexpected metabolites. On the other hand, it is possible that the inert carriers generated from the cleavage of the prodrug are converted into toxic metabolites, such as formaldehyde and acetaldehyde. In some cases, the prodrug may consume important cellular components, such as glutathione, during its activation phase, leading to toxicity. Attention should be paid to the possible new toxicities caused by prodrugs.

Concluding remarks

The prodrug approach is a well-established strategy for addressing pharmaceutical and pharmacokinetic challenges and enhancing the therapeutic efficacy of active drug molecules. It plays an essential role in the screening and optimization of compound structures during drug development. Considering that the structure of a prodrug is different from that of the parent drug, the prodrug should be investigated and evaluated as a new chemical entity, especially for the studies of the physicochemical properties, drug-drug interactions, and the absorption, distribution, metabolism, and excretion of the prodrug in the body.

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Authors: Huijuan Wang, Ruixing Li, Weiqun Cao

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