The Evolution of Prodrug Strategies: From Simple Carriers to Smart, Targeted Systems

Prodrugs are inactive in vitro but become active parent drugs in vivo through enzymatic or chemical metabolism. Prodrugs must undergo controlled or predictable chemical or enzymatic biotransformation into active forms before they can start to have therapeutic effects. (1). When a drug's active ingredient and a "promoiety" form a chemical bond, the term "prodrug" is used to describe the situation. Prodrugs are essential for reducing active medications' negative pharmacodynamic or pharmacokinetic effects.(2). It is a reversible or bio reversible derivative, or a boilable drug-carrier system.(3) Prodrugs are designed for diverse routes of administration, such as oral, transdermal, inhalation, intramuscular, intravenous, etc.(4)

History of Prodrug:

A prodrug is a compound metabolized in vivo before showing its therapeutic effect. The  acetanilide was first prodrug introduced by  Cahn and Hepp in 1867. It was an antipyretic agent (under the name Antifebrin). In vivo, Acetanilide undergoes aromatic hydroxylation into acetaminophen (paracetamol), an active compound with antipyretic and analgesic effects.(5)  The Parke-Davis company first applied the prodrug concept to modify chloramphenicol as chloramphenicol palmitate, to enhance its taste and increase aqueous solubiity.(6)

Ideal Properties of a Prodrug:       

a) Pharmacological inertness in the absence of body fluids.

b) the ability to quickly change into the drug's active form at the site of action.

c) the capacity to produce nontoxic metabolites and eliminate them on their own.⁽⁷⁾

Benefits of Prodrug Approach:

• Improving drug water-solubility⁽⁸⁾
• Improving Chemical Stability
• Reducing Pain and Irritation
• Improving Bioavailability and Oral Permeability
• Improving Site Specificity
• Increasing the Duration of Action.⁽⁹⁾
• Improving the Organoleptic Characteristics.⁽¹⁰⁾

Classification of Prodrug:

Prodrugs Are Divided into Two Main Categories:

1. Carrier-Linked And
2. Bioprecursors Prodrugs.

1. Carrier-Linked Prodrugs:

A prodrug that includes a temporary bond between the active substance and a transient carrier group, enhancing its physicochemical or pharmacokinetic properties, and which can be easily removed in vivo, via hydrolytic cleavage. known as a carrier-linked prodrug(4)

carrier exhibits the properties as follows:

• It keeps the prodrug at the site of action by maintaining its structure until it reaches the target site.
• It is nontoxic molecule and must be biochemically inert.
• Promotes drug release via chemical or enzymatic processes.⁽⁷⁾

It is categorized as follows based on the type of carrier used:

Bipartite, double, or cascade-latentiated prodrug:

In bipartite prodrugs, the drug molecule is joined to a single carrier moiety. The majority of prodrugs attached to carriers are bipartite.⁽⁷⁾. For example, The enzyme human valacyclovirase is responsible for hydrolyzing the antiviral nucleoside prodrug valganciclovir, thereby releasing the active drug ganciclovir.⁽¹¹⁾

Tripartite Prodrug:

In tripartite prodrugs, the carrier is attached to the drug moiety via a linker. for example, Capecitabine is orally absorbed and enzymatically converted in three steps by carboxylesterase, cytidine deaminase, and thymidine phosphorylase into its active metabolite 5-fluorouracil (5-FU), which is employed in the management of breast cancer.(12).

Macromolecular Prodrugs:

These prodrugs are high-molecular-weight complexes containing several drug molecules.

Site-Specific Prodrugs:

These prodrugs increase the effectiveness of drug delivery by directing the active ingredient to a particular site. For example, sulfasalazine is composed of sulfapyridine and 5-aminosalicylic acid  in the treatment of inflammatory bowel  disease(13).

Mutual Prodrugs:

These are combinations of two biologically active substances that work  as a complementary group for each other.(1) For example, Estramustine is a major metabolite of the prodrug estramustine phosphate used in management of prostate carcinoma. it falls under the class of microtubule-targeting agents.(4)

Another example of mutual pro-drugs involves the coupling of ibuprofen (NSAID) with sulfa drugs, such as sulfanilide, sulfacetamide, sulfamethoxazole, and sulfisoxazole are used to overcome the drawback of gastrointestinal irritation and ulceration produced due to the free carboxylic group of ibuprofen. (7)

2. Metabolic Precursor/Bio precursor

This involves the conversion of an inactive drug into an active form through chemical modification, resulting in a compound metabolized by enzymes.(4) Phenylbutazone is a metabolic precursor prodrug of oxyphenbutazone.(4) Phenylbutazone used in the treatment of rheumatic diseases.(14)

Based on their site of conversion into the active drug, they are categorised as
as follows:

1. Type I (metabolized intracellularly) and
2. Type II (metabolised extracellularly).
3. Mixed-Type

1. Type I: Type IA prodrugs (e.g., acyclovir, cyclophosphamide, 5-fluorouracil, L-DOPA, zidovudine) undergo intracellular metabolism.
2.  Type II: These are metabolized extracellularly. These are further classified as,

Type IIA: metabolized in the gastrointestinal fluid (e.g., sulfasalazine, loperamide oxide).

Type IIB: metabolized within the extracellular fluid or circulatory system (e.g., aspirin, bambuterol, fosphenytoin).

Type IIC: metabolized near the therapeutic target. (3,15)

Mixed-Type: These prodrugs undergo multiple conversions within target cells and metabolic tissues, such as simvastatin, a lipid-lowering agent and a classic Type IA prodrug, which is converted in vivo to its active metabolite, simvastatin acid a potent HMG-CoA reductase inhibitor and certain chemotherapy agents such as capecitabine, are converted intracellularly to the active anticancer compound 5-fluorouracil (5-FU). These are typically classified as Type IA/IB prodrugs, due to their intracellular activation.(15)

Rationale for Prodrug Design

• Prodrugs that improved permeability

1. Tenofovir and sofosbuvir are antiviral prodrugs that uses ProTide technology to enhance permeability. Tenofovir, the active drug, is administered as tenofovir disoproxil fumarate or tenofovir alafenamide, which are ester prodrugs. These prodrugs improve lipophilicity, allowing better membrane permeability and oral absorption. Inside cells, they are converted to the active tenofovir diphosphate, which blocks viral reverse transcriptase^.(16)(4)
2. sofosbuvir is a phosphoramidate prodrug of 2'-deoxy-2'-fluoro-2'-C-methyluridine monophosphate, and its phosphoramidate moiety enhances its lipophilicity, allowing better cell membrane penetration. After entering the cell, it is activated to its active triphosphate form, which inhibits HCV RNA polymerase^.(16)
3. Carbecillin and Geocillin are prodrugs designed to improve membrane permeability, enhancing their absorption and bioavailability. These prodrugs are modifications of carbenicillin, a penicillin antibiotic, and are specifically designed to overcome the barriers of poor oral bioavailability and cellular permeability.  

• Increasing the Duration of Action

The fluphenazine ester prodrugs increase patient compliance by lowering the frequency of doses and extending the duration of action. ⁽¹⁷⁾

• Prodrug with improved drug aqueous solubility:

1. Isavuconazonium sulfate, an acyloxyalkyl triazolium salt of the antifungal agent isavuconazole, is used to enhance prodrug solubility.
2. Tedizolid phosphate is another prodrug used for the treatment of acute skin infections, offering improved solubility over its active form.(18).

• Targeted Drug Delivery: -

Gabapentin enacarbil, a prodrug for the treatment of postherpetic neuralgia and restless legs syndrome, is an example of targeted drug delivery. MCT-1 and sodium-dependent multivitamin transporter are two high-capacity nutrient transporters that are substrates of gabapentin enacarbil, a prodrug of gabapentin. (19)

• Enhance the parent drug's pharmacokinetic profile:

Fesoterodine fumarate, which treats overactive bladder syndrome, was created to reduce pharmacokinetic variability between patients. (20)

• Improving the Organoleptic Characteristics

To enhance organoleptic qualities (e.g., chloramphenicol palmitate is a poorly soluble precursor of chloramphenicol, which is converted to the active form by pancreatic lipase and tasteless due to its low aqueous solubility).(21)

Reducing Pain and Irritation

Prodrugs, such as clindamycin palmitate, are designed to improve patient compliance by minimizing pain and irritation during administration.(1)

Improving Chemical Stability:

antineoplastic agents, like azacytidine, are designed to improve chemical stability, ensuring prolonged shelf life and effectiveness.

• Improving Site Specificity

Prodrugs such as capecitabine are designed for site-specific activation, converting into the active drug 5-fluorouracil primarily within tumor tissues due to higher levels of thymidine phosphorylase. This selective activation improves therapeutic outcomes while minimizing systemic side effects.(22)

Prodrug Activation Mechanism

Fatty acid amide hydrolases

The carboxylate-containing sobetirome's prodrug was created as an ethanolamino ester prodrug, which easily undergoes O, N-acyl migration to produce the amides.(23). The N-methyl amide form of sobetirome is the most effective for targeting the central nervous system while minimizing its activation outside the brain.(24)      

Phosphoramidase-catalyzed hydrolysis:

Microsomal enzymes mediate the bioactivation of cyclophosphamide (CPA) in the presence of cofactors like nicotinamide adenine dinucleotide phosphate. The first byproduct of CPA metabolism is 4-hydroxy-CPA, which opens its rings to yield aldophosphamide. Phosphor amide mustard is produced when aldophosphamide breaks down due to a spontaneous β elimination.^{(22 )} Phosphamides, an enzyme that hydrolyses the phosphorus–nitrogen bond and releases the cytotoxic nitrogen mustard, is highly expressed in tumour cells, which is the basis for the chemical design of CPA.(26)

CYP-mediated N-demethylation

dacarbazine used as the first-line treatment for malignant melanoma.(27) Dacarbazine is a prodrug based on triazene that requires bioactivation before its activation. First, dacarbazine is N-demethylated by CYP to produce the reactive species 5-[3-hydroxy methyl-3-methyl-triazen-l-yl]-imidazole-4-carboxamide,. (MTIC) After formaldehyde is lost. MTIC rapidly degrades to generate the highly reactive intermediate methane diazohydroxide, which in turn releases molecular nitrogen and a methyl cation that methylates guanine at the O-6 position, causing significant cytotoxicity, along with the formation of the primary plasma and urinary metabolite aminoimidazole carboxamide (AIC).

Cyclization-Activated Prodrug

O-benzoyl pilocarpic acid benzyl ester is a precursor of pilocarpine designed to enhance ocular delivery. In aqueous solution, it undergoes base-catalyzed hydrolysis, releasing a hydroxyl group. This triggers intramolecular cyclization (lactonization), resulting in the formation of active pilocarpine. The mechanism allows controlled release at the target site.(28) (29)

Prodrugs with disease-specific activation mechanisms:

Certain enzymatically activated prodrugs take advantage of special enzyme activity resulting from the disease they are meant to treat, rather than using patient-specific enzymes for conversion. Benznidazole, used to treat paediatric Chagas disease,contains  prodrug 2-nitroimidazole, which is activated by enzymes called parasite type I nitro reductase.(30)

New Developments in Prodrug Therapies

Prodrug-based Nano-DDS methods for cancer treatment

High drug loading, improved stability, enhanced tumor selectivity, and reduced toxicity are the benefits of prodrug-based Nano-DDS (Nanoscale Drug Delivery Systems), which combine the benefits of prodrugs and nanotechnology into a single nanoplatform for efficient drug delivery.(30). Nanoscale Drug Delivery Systems are used to overcome the difficulties associated with conventional drug delivery.(31)

Prodrug Enzyme-Directed Therapies

It involves a two-step treatment process involving targeted administration of an exogenous enzyme followed by administration of a complementary prodrug. The prodrug is made especially to release the active drug only after interacting with the exogenous enzyme, whereas the enzyme is directly targeted to a desired site in the body using antibodies, polymers, or genes.(30)

Enzyme-responsive small-molecule prodrugs

Enzyme-activated small-molecule prodrugs are engineered to release their active drug exclusively in the presence of certain enzymes that are overproduced in tumors. For example, irinotecan is a prodrug activated by carboxylesterases to release 7-ethyl-10-hydroxycamptothecin, a potent topoisomerase I inhibitor used in cancer therapy. This selective activation allows the drug to work mainly in tumor tissues, reducing systemic toxicity.

Light-Responsive Moieties for Imaging and Therapy.

Light-responsive imaging moieties are incorporated into enzyme-activated prodrugs to visualize drug distribution and monitor activation in vivo. For example, β-galactosidase-activated prodrugs of doxorubicin linked with dyes like fluorescein isothiocyanate, enabling both therapeutic action and fluorescent tracking. Upon enzymatic activation in target tissues (e.g., tumors), the prodrug releases active doxorubicin, while the fluorophore allows non-invasive imaging of drug release.  

Lectin-directed therapy

Lectin-directed therapy is a two-part system that delivers a prodrug with a sugar group and a glycosylated enzyme to cancer cells in a specific manner. It uses lectin, a protein that binds to a sugar molecule. The enzymes used in this process, such as a-rhamnosidase, are found naturally in a particular species.

Applications of Prodrug:

1. Antiviral agents:

Fludarabine phosphate; phosphate ester of fludarabine

The compound is bio converted by alkaline phosphatases into fludarabine, which is then transformed into 2-fluoro-9-β-D-arabinofuranosyladenine. After entering cells, it is further metabolized into the active 2-fluoro-9--D-arabinofuranosyladenine 5’-triphosphate.(3)

Adefovir Dipivoxil

Adefovir dipivoxil, bis-(pivaloyloxymethyl) ester of adenofovir Converted by esterases and phosphodiesterases into tenofovir. Within T lymphocytes, tenofovir is transformed into its active form, tenofovir diphosphate, which acts as an inhibitor of the reverse transcriptase enzyme in the HIV virus (anti-HIV).(3)

Valganciclovir

Valganciclovir is a prodrug of ganciclovir that is an L-valine ester. After oral administration, the valganciclovir is absorbed from the intestine through the peptide transporter PEPT1. The liver and intestinal esterases quickly convert valganciclovir into its active form, ganciclovir. (32) A significant benefit of valganciclovir is that ganciclovir's bioavailability is approximately ten times higher compared to that from oral ganciclovir capsules. (33)

2. Anticancer Agents:

Evofosfamide

Under extremely low oxygen levels (less than 0.5% O2), typical of areas within tumors that are poorly oxygenated, the radical anion variant of the prodrug experiences reductive activation, resulting in the liberation of the active cytotoxic compound, Br-IPM (bromo-isophosphoramide mustard), along with an azole byproduct. The liberated Br-IPM subsequently alkylates DNA, creating in restrain and interstrand cross-links, which interfere with essential cellular functions and trigger the death of tumor cells.(34)

Miproxifene phosphate, TAT-59

Miproxifene phosphate (TAT-59) is a phosphate ester prodrug of miproxifene, developed as an anticancer agent. It is activated in the body by alkaline phosphatase enzymes that are more active in certain tumor cells, releasing the active drug miproxifene, a selective estrogen receptor modulator (SERM) with anti-tumor properties. This enzymatic activation enhances the solubility and bioavailability of the drug for efficient cancer treatment.(17)(35)

Ixazomib

The ester prodrug of Ixazomib, known as ixazomib citrate, is utilized for treating multiple myeloma. The active mechanism of Ixazomib involves reversible inhibition of the beta 5 subunit of the 20S proteasome.

Recently Approved Prodrugs:

Remdesivir:  (Prodrug of R835)

Remdesivir (Veklury): Remdesivir acts as a prodrug for an adenosine nucleotide analogue. It is effective against Ebola, Nipah, respiratory syncytial virus (RSV), SARS-CoV-2.(36). Remdesivir inhibits the RdRp of coronaviruses and demonstrates antiviral activity in both cell cultures and animal models.

Baloxavir marboxil:-

Baloxavir marboxil is a prodrug of the selective PA inhibitor(Polymerase Acidic protein^.(37) It has demonstrated nanomolar antiviral effectiveness against both influenza A and B viruses in vitro, including strains that are resistant to existing antiviral treatments. The active form of this prodrug is baloxavir acid (BXA). Baloxavir marboxil is converted to its active form, baloxavir acid, which binds to CEN(Cap-dependent Endonuclease). In vitro preclinical shows that baloxavir acid halted viral RNA transcription and replication.(38). As an alternative strategy for influenza treatments, baloxavir acid and its prodrug, baloxavir marboxil, were developed, incorporating a phenolic hydroxyl group to improve the oral absorption of baloxavir acid.(39)

Omidenepag Isopropyl: -

Omidenepag isopropyl is used in lowering elevated intraocular pressure to treat open-angle glaucoma.(40). Omidenepag isopropyl (OMDI) acts as a prodrug of omidenepag. It is converted into the active form, omidenepag, through esterase-mediated hydrolysis in the eye as it penetrates the cornea.(41)

Tafenoquine

Tafenoquine, Is Anti-Malarial Agent. It Is Metabolized Into Its Active Form, 5,6 Ortho-Quinone Tafenoquine, Through Cyp2d6.(42) It Can Target All Stages And Species Of Human Malaria Parasites At Therapeutic Doses. The Activation Of Tafenoquine Requires Metabolism By The Cytochrome P450 2d6 Enzyme Found In The Liver Microsomes.(2)

Eplontersen: (Wainua™)

Eplontersen is a ligand-conjugated antisense oligonucleotide designed to inhibit the synthesis of human transthyretin (TTR) protein used for the treatment of TTR-mediated amyloidosis. In the liver, eplontersen is hydrolysed into short oligonucleotide fragments by endo- and exonucleases.(43). Eplontersen functions through a complex mechanism by promoting the degradation of TTR messenger RNA (mRNA) within liver cells (hepatocytes).(44)

Fostemsavir:

Fostemsavir is a phosphonooxymethyl prodrug of timesaver that employs a distinct mechanism of action by binding to the envelope glycoprotein 120 of HIV, preventing the virus from attaching to the CD4 receptors on host cells.(2). FTR shows a favorable pharmacokinetic profile, characterized by slow dissolution and enhanced absorption.(45). This prodrug was developed to improve the solubility of the compound within the gut. The alkaline phosphatase, found on the surface of the small intestine's brush border membranes, cleaves the prodrug.(46).Alkaline phosphatase converts the prodrug into its active form in the gastrointestinal tract just prior to absorption.(47)

Brincidofovir:

(BCV) is an antiviral drug. Its parent compound, cidofovir, was demonstrated in vitro efficacy against the variola virus. its clinical use was limited by a challenging side effect profile, particularly high levels of nephrotoxicity. In humans, Brincidofovir (CMX001) is processed by the liver into three main metabolites: CMX104, cidofovir, and CMX064.(48).The design of CMX001 allows it to remain unaltered in plasma and facilitates direct delivery of the drug to targeted cells. This design enhances cellular uptake and leads to increased intracellular levels of the active antiviral metabolite, cidofovir diphosphate.⁽⁴⁹⁾ In contrast to cidofovir, Brin cidofovir avoids interaction with organic anion transporter 1 in the renal tubules, thereby preventing nephrotoxicity^.(50)