Amino Acids for Prodrug Development

Prodrug refers to a type of compound that, through chemical modification, does not exhibit pharmacological activity upon entering the body. It is converted into an active drug through metabolic processes within the body. Amino acids, as natural organic molecules, have unique structural characteristics and biocompatibility, making them widely applicable in prodrug design. Amino acids not only offer unique advantages in drug design but also enhance the biological activity, pharmacokinetic properties, and targeting of drugs through various chemical modifications.

What is a Prodrug?

A prodrug, also known as a precursor drug, drug precursor, or prodrug compound, is a substance that, after chemical modification, has little or no activity in vitro but becomes an active drug upon metabolic conversion within the body, where it exerts its pharmacological effects. Prodrugs have little or no biological activity until they are metabolized into active compounds within the body. This process aims to improve the drug's bioavailability, enhance targeting, and reduce toxicity and side effects. Generally, prodrugs have the following characteristics:

• Drug-related aspects: Improve solubility, chemical stability, and sensory properties. Reduce local irritation or pain following administration to minimize issues associated with the formulation of active agents.
• Pharmacokinetic aspects: Improve absorption routes (e.g., oral or intravenous injection), reduce metabolism across systems, improve temporal characteristics, and enhance selective delivery of the active agent to tissues or organs.
• Pharmacodynamic aspects: Reduce toxicity and improve the therapeutic index by designing single chemical entities combining two drugs (combination drug strategies).

Prodrug vs Active Drug

A prodrug is a chemical compound that lacks pharmacological activity until it undergoes metabolic conversion in the body to form an active drug. Prodrug design is typically aimed at improving drug bioavailability, solubility, stability, or targeted delivery. Prodrugs are metabolized through enzymatic processes or organ-specific metabolism (e.g., liver), generating the active drug. An active drug refers to a compound that directly exerts therapeutic effects by interacting with targets in the body, such as receptors, enzymes, or other biological molecules. Active drugs usually have well-defined pharmacological effects and mechanisms of action. They exert therapeutic effects by directly binding to their targets without requiring conversion. In summary, a prodrug must undergo metabolic conversion to become an active drug, while an active drug exerts its effects directly. The use of prodrugs can offer significant advantages in enhancing efficacy and reducing side effects.

Example for Prodrug

Currently, approximately 10% of marketed drugs can be classified as prodrugs, and it is expected that more prodrugs will enter the pharmaceutical market in the future. Below are some representative prodrugs:

Types of Prodrugs

In most cases, prodrugs can be structurally designed based on known biotransformations, achieved through chemical modifications or derivatizations of the parent drug. These modifications enable the prodrug to be activated by enzymatic or non-enzymatic processes, releasing the parent drug to exert its pharmacological effect. Based on the structure of the prodrug and its activation mechanism, prodrugs can be divided into two categories: carrier prodrugs and biological prodrugs.

• Carrier Prodrugs

Carrier prodrugs are formed by linking an active drug (parent drug) to a non-toxic, transport-active compound, which is typically lipophilic. Carrier prodrugs have three key characteristics: first, the prodrug itself should have little to no activity, or its activity should be less than that of the parent drug; second, the parent drug is generally covalently bound to the carrier, but upon entering the body, the bond can break to release the parent drug, usually through simple acid or base hydrolysis or enzyme-catalyzed conversion; third, the prodrug must typically release the parent drug at a rapid rate in the body to ensure sufficient concentration of the parent drug at the target site. However, when the modification of the parent drug aims to prolong its action for sustained-release effects, a prodrug with slower metabolic conversion can be designed. The core challenge in designing carrier prodrugs is selecting the appropriate carrier and ensuring that the parent drug is released at the intended site of action, taking into account differences in enzymes, receptors, and pH conditions within different tissues.

• Biological Prodrugs

Biological prodrugs refer to new compounds obtained by modifying the structure of existing active molecules. These new compounds act as substrates for metabolic enzymes, which convert them into the expected active molecules. There are many examples of biological prodrugs, including several blockbuster drugs. For instance, the lipid-lowering drug simvastatin is activated in the body by esterases, which open the six-membered lactone ring to release the active form; the proton pump inhibitor omeprazole undergoes acid-catalyzed Smiles rearrangement in the body, followed by transformation into sulfinic acid and sulfonamide to become active; and the anticoagulant clopidogrel is metabolized by enzymes, then hydrolyzed to open the thienopyridine ring before becoming active. The development of biological prodrugs involves a degree of serendipity, as many active metabolites are discovered after the prodrug has been created, rather than being designed in advance. Due to the complexity of the enzyme systems in the human body, biological prodrugs may undergo diverse transformations, making it challenging to produce the expected active molecules. Therefore, compared to carrier prodrugs, the development of biological prodrugs is more difficult.

Design of Prodrugs

The design of prodrugs involves modifying the structure of the parent drug molecule by adding or altering functional groups to generate a new chemical entity. This new entity often results in changes to the drug's properties, especially when the parent drug has pharmacokinetic shortcomings, such as poor solubility (leading to low bioavailability), rapid metabolism (causing insufficient pharmacological effect), or inadequate targeting, which results in drug toxicity. Through prodrug design and optimization, these issues can be effectively addressed, improving or circumventing the defects of the parent drug.

• Improving Drug Solubility

The drug needs to be dissolved to be taken up. Suboptimal solubility prevents drugs from reaching and travelling throughout the body. The addition of polar functional groups to the drug structure (for example amino acid esters, polyethylene glycol, sugars, or phosphate esters) can help prodrug designs be much more soluble. For example, Degoey et al. formulated a phosphate ester prodrug of lopinavir that solubilised the drug more than 700 times better than free lopinavir and, in pharmacokinetic experiments, showed that the active drug concentration in plasma was higher when it was released from the phosphate prodrug following oral administration.

• Influencing Drug Membrane Permeability

The cross-cell adhesion of a drug is dependent on its lipophilicity and the recognition of the cell membrane transport proteins. The prodrugs could increase lipophilicity by obscuring polar groups or by forming lipophilic groups, or they could make membranes more permeable by adding functional groups recognised by transport proteins. Inhibitor of neuraminidase, oseltamivir, for example, is an ethyl ester prodrug of RO-64-0802, the functional ester group covering the polar carboxylic acid group. Compared to RO-64-0802, oseltamivir is more lipophilic and bioavailability from 5% to 80%.

• Improving Drug Metabolic Stability

In the presence of metabolically sensitive areas in the drug's matrix, resulting in high rates of rapid and extensive metabolism in the liver or gastrointestinal tract is what is called metabolic instability. Such instability can limit the volume of active drug in the body that reaches the bloodstream and interfere with its therapeutic activity. Simply by editing the functional groups in a prodrug to protect these metabolically vulnerable regions, its metabolism can be improved and the active drug protected from pre-systemic metabolism. The prodrug of terbutaline, for instance, bambuterol (a dimethylcarbamoyl ester prodrug of terbutaline), which carries the phenolic hydroxyl group out of the body, limits the amount of first-pass metabolism. Bambuterol is mostly slowly broken down by unreliable butyrylcholinesterase to give us terbutaline which you can take once per day with less side effects than when you take terbutaline three times a day.

• Enhancing Drug Targeting

It is essential for a drug to target the target organ or tissue correctly so it can function as a drug. Changes in structure to promote targeting can augment the drug's therapeutic activity without affecting the adverse effects. Targeting moiety (such as peptides or antibodies) with cytotoxic molecules is often used in protodrug applications. PDCs – in which linkers bind peptides to drugs, to create an effective drug – for instance, can collect selectively on tumour cells, increase half-life and increase efficacy. So do antibody-drug conjugates (ADCs), which are monoclonal antibodies that bind very tightly to antigens on the surface of the tumour cell, allowing receptor-mediated endocytosis and direct delivery of active drugs inside the tumor cells. Prodrug targeting designs include, too, successful plans that attach active anticancer agents to folate or DNA aptamer chains, thus targeting cancer cells.

Amino Acids in Prodrug Design

Amino acids are fundamental components of cellular structures but require specialized transport systems to cross the plasma membrane. Research has identified and classified several of these transport systems based on their substrate affinity, dependence on sodium ions, energy requirements, and pH sensitivity. Amino acids are low-toxicity agents, making them an attractive carrier for developing prodrugs of therapeutic agents with poor absorption. Furthermore, amino acid prodrugs can enhance the water solubility of the parent drug. The importance of these transport proteins in pharmacokinetics has been recognized in several studies, which report improved bioavailability of amino acid-conjugated compounds.

Fig. 1. Amino acids in prodrug design and development (Adv Drug Deliv Rev. 2013, 65(10): 1370-85).

Esterification reactions are one of the most widely applied strategies in prodrug design. The carboxyl group of an amino acid can form an ester with the hydroxyl group of the parent drug, while the amino group can form a salt with an inorganic acid, thereby increasing the drug's water solubility. For example, metronidazole N,N-dimethylglycine ester hydrochloride has good water solubility and high plasma concentration. However, aqueous solutions of this prodrug are unstable and must be prepared before use. This instability is due to the protonation of the amino group at a pH of 3–5, which has a strong electron-withdrawing effect, activating the ester carbonyl group and making it susceptible to attack by OH- ions, causing ester bond cleavage. Studies have shown that introducing a phenyl group between the ester and amino groups to form N-substituted amine methylbenzoate can completely prevent the amino group from affecting the ester bond while not interfering with enzymatic hydrolysis in the body. This modification increases the water solubility and stability of the prodrug solution, which can remain stable for up to 14 years under the same conditions.

Amino Acid from BOC Sciences

BOC Sciences has strong capabilities in the supply of amino acids and offers a wide range of standard and non-standard amino acid products. We provide high-quality amino acids to the global pharmaceutical, food, chemical, and biotechnology industries, with a particular focus on prodrug development. Our custom amino acid synthesis services include various amino acid derivatives for use in drug delivery, protein engineering, and peptide drug development. We leverage advanced synthetic technologies and an experienced team of experts to develop specific amino acid derivatives according to customer needs, supporting applications in prodrugs, targeted therapies, and biopharmaceuticals. Additionally, we offer stringent quality control to ensure that all products meet cGMP standards, guaranteeing product stability and consistency. Whether for small-scale custom orders or large-scale production, BOC Sciences provides flexible and efficient solutions for our clients.

What is the Use of Amino Acids in Prodrug Development?

Amino acids, when conjugated with drug molecules, can enhance the bioavailability and stability of the drugs. Amino acid derivatives can be enzymatically converted into active drugs in the body, improving drug targeting, especially in the treatment of tumors and infections. Moreover, the hydrophilicity and water solubility of amino acids contribute to drug absorption and distribution, improving pharmacokinetic properties, extending drug half-life, and reducing toxicity in non-target sites. Prodrugs derived from amino acids can also minimize drug toxicity to normal cells during conversion in the intestine or liver. For instance, anticancer and antimicrobial drugs modified with amino acids significantly improve efficacy and reduce side effects by releasing active drugs at target cells. The practical applications of amino acids in prodrug design mainly include:

• Amino Acid-Derived Anticancer Drugs

Anticancer drugs are primarily cytotoxic agents that exert anti-tumor activity by interfering with aspects of DNA replication, repair, translation, or cell division. However, they do not only destroy tumor cells; they can also harm normal cells, leading to severe adverse reactions. Therefore, the main goal of prodrug strategies for anticancer drugs is to reduce their toxicity. One effective and attractive approach to lowering toxicity is the design of prodrugs that are selectively activated in target tissues. To achieve effective targeting, enzymes or transport proteins that are specifically or preferentially expressed in the target tissue are required, which is a significant challenge. Despite this, several prodrug strategies have been employed to improve solubility, transport, and pharmacokinetic characteristics. For example, the successful outcomes of valacyclovir and valganciclovir suggest a great potential for amino acids as components of other drug prodrugs. This success is attributed to their enhanced intestinal transport through oligopeptide transporters. In fact, amino acid ester prodrugs significantly increase cellular absorption of the parent drug through peptide transport mechanisms, even though they do not contain peptide bonds in their structure.

• Amino Acid-Derived Antiviral Drugs

Similarly, amino acid-derived antiviral drugs also exhibit good prodrug characteristics, as they can be converted into active forms by enzymes at the site of viral infection, targeting specific viral markers for treatment. For example, the oral bioavailability of the antiviral drug acyclovir is poor due to a lack of transport systems in the intestine that can recognize the drug as a substrate. To improve this characteristic, several prodrugs of acyclovir have been synthesized. Different methods have been employed to achieve this goal. Researchers such as Colla and Maudgal have used amino acid components to prepare acyclovir prodrugs with potential applications in the formulation of eye drops. Beauchamp and others adopted similar methods to improve oral administration. For various purposes, Shao and Yang have reported the synthesis, nasal and ocular absorption, and metabolism of fatty acid ester prodrugs of acyclovir. Recently, Gao and Mitra have focused on improving membrane transport of acyclovir prodrugs. Their research led to the development of a series of N-acyl-ACV, α, β, and γ-amino acid esters and dicarboxylates of acyclovir.

• Amino Acid-Derived Antimicrobial Drugs

The application of amino acids in prodrug design is also relevant to the development of antimicrobial drugs. Some prodrug molecules of antimicrobial agents, modified with amino acids, can enhance drug stability and targeting. These drugs often release their active ingredients at specific sites through enzymatic reactions in the body, reducing toxicity to non-target cells. For instance, Ibrahimi and colleagues synthesized amino acid conjugates of fluoroquinolones, ciprofloxacin, norfloxacin, metronidazole, and sulfamethoxazole and screened them for activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. All the drugs were found to be broad-spectrum antibiotics with activity against both Gram-positive and Gram-negative bacteria.

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