Synthetic Prodrug Research Articles

Exploring Biological Targets of Magnolol and Honokiol and their Nature-Inspired Synthetic Derivatives: In Silico Identification and Experimental Validation of Estrogen Receptors.

In this work, we describe the results of a computational investigation aimed at identifying potential biological targets of honokiol, magnolol and a series of synthetic prodrug derivatives obtained through esterification of the free hydroxyl groups. The ligand-based and structure-based analyses revealed that these compounds potentially interact with several biological targets, some of which are known while others are new. Honokiol, magnolol, and three of the newly synthesized derivatives may bind to estrogen receptors ERα and ERβ. Biological testing confirmed that these compounds modulate estrogen-regulated transcriptional activity mediated by ERα or ERβ with potencies in the nanomolar range. In particular, magnolol and one of its derivatives (10) behaved as partial antagonists of ERα and ERβ, while compounds 8 and 11 behaved as partial agonists. These findings validate the computational predictions and shed light on the mechanism of action of these natural compounds, paving the way for further investigation in the context of targeted therapies.

Bempedoic acid – new agent in hiperlipidemia treatment – literature review

Hyperlipidemia is becoming more and more severe condition among modern society. High-fat diet, sedentary lifestyle, lack of physical activity are the common factors leading to overweight, obesity and problems with serum lipid control. Dyslipidemia is one of the most important agents contributing to cardiovascular diseases snd therefore lower quality of life, disability and preamature death. This forces to constatnt searching for lipid-lowering drugs, that can be successfully use in the patients suffering from poor lipid control. Bempedoic acid (ETC-1020) is one of the newest, first in the class medicine, firstly approved to use in the USA on 21st February 2020 in monotheraphy, and then on 26th February in combination with ezetimibe. It is a small synthetic prodrug molecule that is activated by acyl-CoA synthetase in the liver. Bempedoic acid inhibits competitively adenosine triphosphate citrate lyase (ACL) and as a result it stops the further production of cholesterol and fatty acids. Research shows the ability of ETC-1020 to decline of LDL-C cholesterol up to 24%. It is also responsible for total cholesterol, non-HDL-C, ApoB and hs-CRP reduction. Bempedoic acid is characterized by good pharmacokinetics and pharmacodynamics. ECT-1020 has also a good drug-to-drug interaction profile. Many clinical trials have reported efficacy and safety of use with minimal side effects. Because of its target point is located in the liver and kidney, it can be successfully use in patients with statin intolerance due to bypassing decline of muscle cholesterol biosyntheisis, that is the main cause of musculosketelar adverse effects.

Fine-tuning the activation behaviors of ternary modular cabazitaxel prodrugs for efficient and on-target oral anti-cancer therapy

The disulfide bond plays a crucial role in the design of anti-tumor prodrugs due to its exceptional tumor-specific redox responsiveness. However, premature breaking of disulfide bonds is triggered by small amounts of reducing substances (e.g., ascorbic acid, glutathione, uric acid and tea polyphenols) in the systemic circulation. This may lead to toxicity, particularly in oral prodrugs that require more frequent and high-dose treatments. Fine-tuning the activation kinetics of these prodrugs is a promising prospect for more efficient on-target cancer therapies. In this study, disulfide, steric disulfide, and ester bonds were used to bridge cabazitaxel (CTX) to an intestinal lymph vessel-directed triglyceride (TG) module. Then, synthetic prodrugs were efficiently incorporated into self-nanoemulsifying drug delivery system (corn oil and Maisine CC were used as the oil phase and Cremophor EL as the surfactant). All three prodrugs had excellent gastric stability and intestinal permeability. The oral bioavailability of the disulfide bond-based prodrugs (CTX-(C)S-(C)S-TG and CTX-S-S-TG) was 11.5- and 19.1-fold higher than that of the CTX solution, respectively, demonstrating good oral delivery efficiency. However, the excessive reduction sensitivity of the disulfide bond resulted in lower plasma stability and safety of CTX-S-S-TG than that of CTX-(C)S-(C)S-TG. Moreover, introducing steric hindrance into disulfide bonds could also modulate drug release and cytotoxicity, significantly improving the anti-tumor activity even compared to that of intravenous CTX solution at half dosage while minimizing off-target adverse effects. Our findings provide insights into the design and fine-tuning of different disulfide bond-based linkers, which may help identify oral prodrugs with more potent therapeutic efficacy and safety for cancer therapy.

Triple coumarin-based 5-fluorouracil prodrugs, their synthesis, characterization, and release kinetics

5-Fluorouracil (5-FU) has been commonly used as a chemotherapy medication in cancer management. However, owing to its substantial side effects, limited selectivity, and tumor resistance, therapeutic usage of 5-FU has been called into doubt. In order to alter the physicochemical properties of 5-FU and improve its targeting, a coumarin-based-esterase-sensitive prodrug system was used to create nine 5-FU prodrugs, which were divided into three series of compounds, A, B, and C. The chemical structures of these prodrugs are confirmed by using FTIR, 1H-NMR, 13C-NMR, and mass spectrophotometers. The chemical stability of these prodrugs in simulated gastric and intestinal fluids as well as their release in human serum were measured spectrophotometrically. The outcomes showed that the synthetic prodrugs had extraordinary stability in the two stimulated fluids with relatively high half-lives, indicating that they can transit through the upper GIT portions without being harmed. The half-lives of the produced prodrugs in human serum vary from 391.30 to 517.94 min, demonstrating their ability to enter their target tissues undamaged and release their active components in pseudo-first-order kinetics. These components include 5-FU, coumarin or one of its derivatives, and one of the 5-FU biochemical modifiers, which are legumain-specific tripeptide, dichloroacetate, or leucovorin. In conclusion, It is hypothesized that the prepared triple coumarin-based 5-FU prodrugs offer several advantages, including improving the physicochemical properties of the parent drug, potentiating its anticancer activity, minimizing its toxic effects, boosting its oral bioavailability, and providing the capacity for specific targeting.

Exosomes as efficient platforms for delivering adenosine-tetra peptide conjugate to pancreatic cancer cells: An in vitro/in silico study

Pancreatic cancer is a fatal malignancy due to poor response to chemotherapeutic treatments. Although adenosine has demonstrated promising cytotoxic effects, its low half-life has restricted its use in cancer treatment. The tetra peptide of Gly-Phe-Leu-Gly is a cathepsin B substrate, and its conjugation to adenosine may produce a prodrug with fewer adverse effects. This study aimed to prepare a tetra peptide prodrug of adenosine and encapsulate it in two types of cell-derived exosomes. A synthetic prodrug of adenosine was prepared with N-substitution of Gly-Phe-Leu-Gly. Exosomes were isolated from AsPC-1 or RAW 264.7 cell lines. The drug or prodrug loading into exosomes and their release profiles were assessed in PBS at pH 7.4. The exosomes' cellular uptake was analyzed by flow cytometry method. MTT assay and Annexin V/FITC kit was used to measure cytotoxicity and apoptotic profile on the pancreatic cancer cells, respectively. An in silico study by molecular docking simulations and ligand‐binding energy evaluation was also done to study binding affinity of adenosine-tetrapeptide prodrug towards Cathepsin B active site. Exosomes with particle sizes of 100–160 nm were shed more efficiently by RAW 264.7 cell line. Drug loading in exosomes was efficient (∼42 %), and the release profile was sustained for more than 24 h. Cellular uptake was higher in autologous exosomes. AsPC-1-exosomes encapsulating prodrug formulation showed higher cytotoxicity, higher cellular uptake, and more efficacious apoptosis. compared to the free prodrug. Gratifyingly, results of in silico analysis perfectly corroborated the potential binding affinity of adenosine-tetrapeptide prodrug towards Cathepsin B active site with improved energy indices compared to free tetra-peptide.

Fluorimetric Determination of Antiviral (COVID-19) Drug Favipiravir in Bulk and Pharmaceutical Dosage Forms

Favipiravir is a synthetic prodrug, which was first discovered while assessing the antiviral activity of chemical agents active against the influenza virus in the chemical library of Toyoma chemicals. It works by inhibiting RNA dependant RNA polymerase (RdRP), an enzyme required for RNA viral replication inside human cells. A simple, rapid, and economic method was developed for the quantitative determination of Favipiravirusing spectrofluorometer. The Favipiravir standard drug solution and sample tablet solution was prepared using double distilled water as a diluent. The different concentrations ofpure drugin the range 2-10 μg/ml and one sample solution were measured for the intensity at 432nm in the spectrofluorometer. The calibration curve was plotted and the sample’s unknown concentration was calculated from the plot. The calibration curve was found to be linear with r2 value obtained as 0.99.There are various other methods available for the quantification of Favipiravir which include RP-HPLC, UV-spectroscopic methods, FTIR, LC-MS with different extraction methods spiked in human plasma but not by using spectrofluorometer. Favipiravir shows fluorescence when dissolved in appropriate solvent hencethis method was developed to quantify Favipiravir which is a simple and efficient method. This method developed is easy and can be used for routine quality control test for Favipiravir pharmaceutical formulations.
 Keywords: Favipiravir, spectrofluorometer, Calibration curve.

Development and Validation of HPLC-UV Method for the Determination of Favipiravir in Human Plasma

Introduction. Coronavirus disease (COVID-19) is an acute infectious disease caused by SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2). Favipiravir is a synthetic prodrug with antiviral activity used for the treatment of COVID-19. There are oral and parenteral dosage forms of favipiravir. Compared with oral administration, parenteral administration has some advantages. Developing a method for the determination of favipiravir in human blood plasma is necessary for performing the analytical part of clinical studies of favipiravir for parenteral administration as an infusion, studying pharmacokinetics, and choosing the optimal dosage of the drug.Aim. The aim of this study is to develop and validate a method for quantitative determination of favipiravir in human plasma by high-performance liquid chromatography with ultraviolet detection (HPLC-UV) for pharmacokinetic studies.Materials and methods. Determination of favipiravir in human plasma by HPLC-UV. The UV detection was set at 323 ± 2 nm. The samples were processed by methanol protein precipitation. Internal standard: raltegravir. Mobile phase: 0.1 % formic acid in water with 0.08 % aqueous ammonia (eluent A), 0.1 % formic acid in acetonitrile with 0.08 % aqueous ammonia (eluent B). Column: Phenomenex Kinetex®, C18, 150 × 4.6 mm, 5 μm. Analytical range: 0.25–200.00 μg/mL.Results and discussion. This method was validated by selectivity, calibration curve, accuracy, precision, spike recovery, the lower limit of quantification, carry-over effect and stability.Conclusion. We developed and validated the method of quantitative determination of favipiravir in human plasma by HPLC-UV. The analytical range was 0.25–200.00 μg/mL in human plasma. The method could be applied in pharmacokinetics studies of favipiravir.

Development of visible spectrophotometric methods for the analysis of favipiravir in pure drug and tablet formulation

Favipiravir is a synthetic prodrug, first discovered while assessing the antiviral activity of chemical agents active against the influenza virus. The present study is aimed at developing and validating a new colorimetric methods for its determination in both pure form and in tablet formulation. The developed method utilizes ion-pair spectrophotometry and is employed for the quantitative evaluation of the drug using the acidic dyes - methyl orange (MO) and methyl red (MR) as ion-pairing agent. The ion-pair complex of drugs with both dyes obeyed Beer’s law in the range of 10 -50 µg/ml with a acceptable correlation coefficients. Recovery by the method was also good, with a relative standard deviation (%RSD) less than 2.5%. The developed method was successfully applied to determine favipiravir in tablet formulation and was demonstrated to be accurate, precise and reproducible. It is also sufficiently sensitive and specific for the determination of the drug in bulk and formulation with satisfactory results.

Synthetic prodrug design enables biocatalytic activation in mice to elicit tumor growth suppression

Considering the intrinsic toxicities of transition metals, their incorporation into drug therapies must operate at minimal amounts while ensuring adequate catalytic activity within complex biological systems. As a way to address this issue, this study investigates the design of synthetic prodrugs that are not only tuned to be harmless, but can be robustly transformed in vivo to reach therapeutically relevant levels. To accomplish this, retrosynthetic prodrug design highlights the potential of naphthylcombretastatin-based prodrugs, which form highly active cytostatic agents via sequential ring-closing metathesis and aromatization. Structural adjustments will also be done to improve aspects related to catalytic reactivity, intrinsic bioactivity, and hydrolytic stability. The developed prodrug therapy is found to possess excellent anticancer activities in cell-based assays. Furthermore, in vivo activation by intravenously administered glycosylated artificial metalloenzymes can also induce significant reduction of implanted tumor growth in mice.

MicroRNAs as Biomarkers in Canine Osteosarcoma: A New Future?

Sarcomas are frequent in dogs and canine species are excellent animal models for studying the human counterpart. However, osteosarcomas are a rare form of sarcoma with high death rates in humans and dogs. miRNAs are small endogenous RNAs that regulate gene expression post-transcriptionally. The discovery of miRNAs could give a contribute in the diagnosis and prognosis of different types of tumors in animal species, as already in humans. The differentiated expression of miRNAs is a frequent finding in cancers and is related to their pathogenesis in many cases. Most canine and human sarcomas show similar miRNA aberrations. Lower levels of miR-1 and miR-133b in canine osteosarcoma tissues were found to increase tumorigenesis through a higher expression of their target genes MET and MCL1. The overexpression of miR-9 promotes a metastatic phenotype in canine osteosarcomas and its capacity as a prognostic biomarker for the disease is currently being evaluated. MicroRNAs at the 14q32 locus could be used as prognostic biomarkers, since their decreased expression has been associated with poor prognosis in canine and human osteosarcomas. Furthermore, a decreased expression of miR-34a in osteosarcoma tumour cells has been associated with shorter disease-free survival times and its reintroduction as a synthetic prodrug shows good potential as a novel therapeutic target to fight the disease. Circulating miR-214 and miR-126 are significantly increased in a broad-spectrum cancer and have the ability to successfully predict the prognosis of dogs. However, further studies are needed to make the use of miRNAs as biomarkers a common practice.

Synthesis, bioconversion, pharmacokinetic and pharmacodynamic evaluation of N-isopropyl-oxy-carbonyloxymethyl prodrugs of CZh-226, a potent and selective PAK4 inhibitor

We have previously disclosed compound 3 (CZh-226), a potent and selective PAK4 inhibitor, but its development was delayed due to poor oral pharmacokinetics. In an attempt to improve this issue, we synthesised a series of prodrugs by masking its terminal nitrogen of the piperazine moiety. Most synthesised prodrugs of 3 have low or no inhibition of PAK4 activity. The stability of synthetic prodrugs was evaluated in PBS, SGF, SIF, rat plasma and liver S9 fraction. Of these, prodrug 19 was not only stable under both acidic and neutral conditions but also could be quickly converted to parent drug 3 in rat plasma and liver S9 fraction. Such effective conversion into parent drug 3 was observed in rats, providing higher exposure of 3 compared to its direct administration. When given via oral route at daily doses of 25 and 50 mg/kg, the prodrug 19 was effective and well tolerated in mouse model of HCT-116 and B16F10.

New Generation Nanomedicines Constructed from Self-Assembling Small-Molecule Prodrugs Alleviate Cancer Drug Toxicity.

The therapeutic index for chemotherapeutic drugs is determined in part by systemic toxicity, so strategies for dose intensification to improve efficacy must also address tolerability. In addressing this issue, we have investigated a novel combinatorial strategy of reconstructing a drug molecule and using sequential drug-induced nanoassembly to fabricate supramolecular nanomedicines (SNM). Using cabazitaxel as a target agent, we established that individual synthetic prodrugs tethered with polyunsaturated fatty acids were capable of recapitulating self-assembly behavior independent of exogenous excipients. The resulting SNM could be further refined by PEGylation with amphiphilic copolymers suitable for preclinical studies. Among these cabazitaxel derivatives, docosahexaenoic acid-derived compound 1 retained high antiproliferative activity. SNM assembled with compound 1 displayed an unexpected enhancement of tolerability in animals along with effective therapeutic efficacy in a mouse xenograft model of human cancer, compared with free drug administered in its clinical formulation. Overall, our studies showed how attaching flexible lipid chains to a hydrophobic and highly toxic anticancer drug can convert it to a systemic self-deliverable nanotherapy, preserving its pharmacologic efficacy while improving its safety profile. Cancer Res; 77(24); 6963-74. ©2017 AACR.

The Hydrolysis of Diclofenac Esters: Synthetic Prodrug Building Blocks for Biodegradable Drug–Polymer Conjugates

Degradation reactions on diclofenac-monoglycerides (3a,b), diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c), diclofenac (1), and diclofenac lactam (4) were performed at 37°C in isotonic buffer solutions (apparent pH range 1-8) containing varying concentrations of acetonitrile (ACN). The concentration remaining of each analyte was measured versus time. Diclofenac-monoglycerides and diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c) were both found to undergo facile and complete hydrolysis in pH 7.4 isotonic phosphate buffer/10% ACN. Under mildly acidic, neutral or alkaline conditions, diclofenac-(p-hydroxybenzoate)-2-monoglyceride (3c) had the fastest hydrolysis rate (t1/2 = 3.23 h at pH 7.4), with simultaneous formation of diclofenac lactam (4) and diclofenac (1). Diclofenac-monoglycerides (3a,b) hydrolyzed more slowly under the same conditions, to again yield both diclofenac (1) and diclofenac lactam (4). There was also transesterification of diclofenac-2-monoglyceride (3b) to its regioisomer, diclofenac-1-monoglyceride (3a) across the pH range. Diclofenac was shown to be stable in neutral or alkaline conditions but cyclized to form the lactam (4) in acidic conditions. Conversely, the lactam (4) was stable under acidic conditions but was converted to an unknown species under alkaline or neutral conditions.

Waterborne beclomethasone dipropionate affects the physiology of fish while its metabolite beclomethasone is not taken up

Asthma is commonly treated with inhalable glucocorticosteroids, including beclomethasone dipropionate (BDP). This is a synthetic prodrug which is metabolized to the more active monopropionate (BMP) and free beclomethasone in humans. To evaluate potential effects of residual drugs on fish, we conducted a 14day flow-through exposure experiment with BDP and beclomethasone using rainbow trout, and analyzed effects on plasma glucose, hepatic glutathione and catalase activity together with water and body concentrations of the BDP, BMP and beclomethasone. We also analyzed hepatic gene expression in BDP-exposed fish by microarray and quantitative PCR. Beclomethasone (up to 0.65μg/L) was not taken up in the fish while BDP (0.65 and 0.07μg/L) resulted in accumulation of both beclomethasone, BMP and BDP in plasma, reaching levels up to those found in humans during therapy. Accordingly, exposure to 0.65μg/L of BDP significantly increased blood glucose as well as oxidized glutathione levels and catalase activity in the liver. Exposure to beclomethasone or the low concentration of BDP had no effect on these endpoints. Both exposure concentrations of BDP resulted in significantly higher transcript abundance of phosphoenolpyruvate carboxykinase involved in gluconeogenesis, and of genes involved in immune responses. As only the rapidly metabolized prodrug was potent in fish, the environmental risks associated with the use of BDP are probably small. However, the observed physiological effects in fish of BDP at plasma concentrations known to affect human physiology provides valuable input to the development of read-across approaches in the identification of pharmaceuticals of environmental concern.

Bioengineering Anabolic Vitamin D-25-Hydroxylase Activity into the Human Vitamin D Catabolic Enzyme, Cytochrome P450 CYP24A1, by a V391L Mutation

CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) to different products: calcitroic acid or 1α,25-(OH)(2)D(3)-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D(3), the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D(3) (1α-OH-D(3)), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)(2)D(3)-24-hydroxylase into an anabolic 1α-OH-D(3)-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)(2)D(3). Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)(2)D(3) via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)(2)D(3) from 1α-OH-D(3), but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D(3) is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D(3) above the heme for hydroxylation.

Trace Determination of Steroids Causing Age-Related Diseases Using LC/MS Combined with Detection-Oriented Derivatization

With the rapid shift to an aging society in Japan, age-related diseases, such as osteoporosis, dementia and cancer, are sharply increasing. The measurement of steroids related to these diseases in biological fluids and tissues is useful for elucidation of the nature, diagnosis and treatment of such diseases. LC/MS is considered to be the most promising method for this purpose due to its specificity and versatility, but it sometimes does not demonstrate the required sensitivity for trace amounts of steroids, because steroids have a rather low response using either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). To overcome this problem, the author developed detection-oriented derivatization procedures for steroids in LC/MS. For ESI-MS, introduction of a permanently charged moiety is effective. Based on this, 2-hydrazino-1-methylpyridine was developed and used in monitoring prostatic 5alpha-dihydrotestosterone, a good index for the follow-up of patients affected by prostate cancer under androgen deprivation therapy and salivary dehydroepiandrosterone, which is now often designated as an anti-aging hormone. A proton-affinitive Cookson-type reagent, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione, was used for the determination of 1alpha-hydroxyvitamin D3 [1alpha(OH)D3], a synthetic prodrug of the active form of vitamin D3, in human plasma, and this new LC/positive-APCI-MS method enabled the pharmacokinetic study of 1alpha(OH)D3 in humans. Electron-capture APCI-MS based on derivatization with 2-nitro-4-trifluoromethylphenylhydrazine was used for the analysis of neurosteroids, which affect brain excitability through action at the neurotransmitter receptors. With this method, the stress-induced rapid biosynthesis of pregnane-type neurosteroids in rat brains was demonstrated.

Isophosphoramide Mustard-Lysine (ZIO-201): Active Moiety of Ifosfamide (IFOS) without Kidney Toxicity.

Ifosfamide (IFOS) and cyclophosphamide (CPA) are widely used anti-cancer drugs. Both are pro-drugs which must be metabolized before their metabolites cross-link DNA. Use of these drugs, especially high-dose IFOS, is complicated by substantial adverse effects of metabolites not directly involved in DNA-cross-linking including kidney, bladder and CNS toxicities. Recently, a lysine-stabilized form of isophosphoramide mustard (IPM-lysine; ZIO-201), an IFOS-metabolite, was developed. ZIO-201 directly cross-links DNA and is active against human cancer cell lines and in mice with human cancer xenografts. To evaluate nephrotoxicity of ZIO-201 we used an in vitro primary rabbit kidney proximal tubule (RPT) cell culture system, which retains many of the characteristics of renal proximal tubule cells including a polarized morphology, a Na+/glucose cotransport system and a p-aminohippurate transport system, glutathione status, and hormone responses (including a parathyroid hormone sensitive adenylate cyclase). We studied effects of ZIO-201 and other IFOS metabolites on primary RPT cells. IFOS is activated by the cytochrome P450 system producing a mixture of 2- and 3-dechloroethylifosfamide and resulting in formation of chloroacetaldehyde (CAA). Also, metabolism of IFOS by the cytochrome P450 system produces 4-hydroxyifosfamide, ultimately resulting in the production of acrolein (ACR). CAA and ACR are IFOS metabolites implicated in kidney toxicity in humans. Effects of ZIO-201 and CAA on the viability of confluent monolayers of primary rabbit kidney proximal tubule (RPT) cells were tested at 15–100 μM. Viability was determined by neutral red dye uptake by the primary cultures after 8 h incubation. ZIO-201 did not significantly reduce viability of primary RPT cells; the LD50 for CAA was about 40 μM with complete inhibition at 75 μM. Effects of 4-hydroperoxyifosfamide (HIFA), a synthetic prodrug of 4-hydroxyifosfamide and ACR were also studied. Although ACR had an LD50 of 80 μM HIFA did not significantly affect viability. The IFOS metabolite 4-hydroxyifosfamide, was prepared from 4-hydroperoxyifosfamide (1). At 200 μM, 4-hydroxyifosfamide resulted in extensive cell death after 4 h incubation. These data show that ZIO-201 avoids the kidney toxicity caused by metabolites of pro-drugs like IFOS. Whereas 2 IFOS metabolites, CAA and ACR were cytotoxic to kidney cells, ZIO-201 was not. An explanation for the toxic effects of 200 μM 4-hydroxyifosfamide t might be metabolism to ACR in vitro.

Water-soluble prodrugs of dipeptide HIV protease inhibitors based on O→N intramolecular acyl migration: Design, synthesis and kinetic study

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-727, a potent small-sized dipeptide-type HIV-1 protease inhibitor consisting of an Apns-Dmt core (Apns; allophenylnorstatine, Dmt; (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid) as inhibitory machinery. These prodrugs contained an O-acyl peptidomimetic structure with an ionized amino group leading to an increase in water-solubility, and were designed to regenerate the corresponding parent drugs based on the O→N intramolecular acyl migration reaction via a five-membered ring intermediate at the α-hydroxy-β-amino acid residue, that is Apns. The synthetic prodrug 3a improved the water-solubility (13 mg/mL) more than 8000-fold in comparison with the parent compound, which is the practically acceptable value as water-soluble drug. Furthermore, to understand the structural effects of the O-acyl moiety on the migration rate, we evaluated several phenylacetyl-type and benzoyl-type prodrugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly under aqueous conditions from slightly acidic to basic pH at 37°C.

New water-soluble prodrugs of HIV protease inhibitors based on O→ N intramolecular acyl migration

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-272 and KNI-279 which are potent HIV-1 protease inhibitors consisting of an Apns–Thz core structure (Apns; allophenylnorstatine, Thz; thiazolidine-4-carboxylic acid) as an inhibitory machinery. The prodrugs, which contained an O-acyl peptidomimetic structure with an ionized amino group leading to the increase of water-solubility, were designed to regenerate the corresponding parent drugs based on the O→ N intramolecular acyl migration reaction at the α-hydroxy-β-amino acid residue, that is allophenylnorstatine. The synthetic prodrugs 3, 4, 6, and 7 improved the water-solubility (>300 mg/mL) more than 4000-fold in comparison with the parent compounds, which is the practically acceptable value as water-soluble drugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly in the aqueous condition from slightly acidic to basic pH at 37 °C, with the suitable migration rate, via a five-membered ring intermediate. Using a similar method, we synthesized a prodrug ( 12) of ritonavir, a clinically useful HIV-1 protease inhibitor as an anti-AIDS drug. In contrast to the prodrugs 3, 4, 6, and 7, the prodrug 12 was very slowly converted to ritonavir probably through a six-membered ring intermediate, with the t 1/2 value of 32 h that may not be suitable for practical use.

Metabolic activation of 1 α-hydroxyvitamin D 3 in human liver microsomes

1. 1α -Hydroxyvitamin D 3 (1 α-OH-D 3) is a synthetic prodrug of the active form of vitamin D 3, and requires the hydroxylation at the C-25 position before eliciting its biological activity. 2. 25-Hydroxylation activities for 1 α-OH-D 3 were present in both microsomal and mitochondrial fractions of human liver. 3. To determine the P450 enzyme(s) involved in microsomal 25-hydroxylation, 14 P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Val, 2D6- Met, 2E1, 3A4, 4A11) were tested for their 25-hydroxylation activity of 1 α-OH-D 3. None catalysed the 25-hydroxylation reaction. 4. 1 α-OH-D 3 in a high concentration (2.5 ng ml -1) showed small but significant inhibition of the catalytic activities of CYP2C8, 2C9-Cys, 2C19, 2D6-Val and 2E1 for their typical substrates. However, 1 α-OH-D 3 in a clinically used low concentration will not significantly affect drug metabolism catalysed by the 14 P450s tested. 5. In summary, the 25-hydroxylation activity of 1 α-OH-D 3 that localizes in the microsomal fraction appears to be attributable to a cytochrome P450 other than the microsomal forms tested in this study.