Degradation Of Prodrug Research Articles

Enzymatic prodrug degradation in the fasted and fed small intestine: In vitro studies and interindividual variability in human aspirates

The aim of the current study was (1) to develop an automation-based protocol for in vitro assessment of enzymatic drug stability at fasted- and fed-state intestinal conditions, (2) to characterize the inter-individual variability of drug degradation in fasted- and fed-state human intestinal fluids, and (3) to compare the obtained in vitro results to drug degradation in human intestinal fluids by taking variability into account. In human intestinal fluids, drug degradation displayed large inter-individual variability, with coefficients of variance generally ranging between 30 and 70 %. The effect of food on the inter-individual variability was highly dependent on the type of drug. The increase of pH in the range between 5.0 and 7.0 significantly accelerated the degradation rate of the studied drugs both in the in vitro and ex vivo experiments. In contrast, the increase of bile salt and phospholipid concentrations in the in vitro screen decreased strongly the degradation rate of the hydrophobic drugs. The developed automated in vitro screen mimicked relatively well the ex vivo degradation of all drugs in the fasted state, whereas in the fed state the degradation of only one of the drugs was adequately reproduced.

Visual evaluation of acetylcholinesterase inhibition by an easy-to-operate assay based on N-doped carbon nanozyme with high stability and oxidase-like activity.

Acetylcholinesterase (AChE) is the key enzyme associated with neurotransmission, and thus many drugs have been explored for their inhibitory effect on AChE, such as donepezil for Alzheimer's disease and organophosphorus pesticides (OPs). Compared with clinical trials, in vitro screening bioassays for AChE inhibitors are preferable in terms of operability and cost. Herein, we developed an easy-to-operate nanozyme-based colorimetric assay for the evaluation of AChE inhibitory strength with excellent anti-interference ability and low dependence on professional equipment. The metal-free carbon nanozyme NC900 played an important role in the signal output due to its features of efficient oxidase-like activity, excellent water dispersibility, high stability and low color interference. Employing various AChE-targeted or non-targeted pesticides as examples, the as-proposed assay exhibited excellent distinguishing ability for different chemicals. The higher absorption intensity at 652 nm represents a stronger inhibitory effect, as well as blue color. In addition, this method was used to study the influence of pH on the degradation of prodrugs, and the efficiency of mixed pesticides. This work provides a simple and reliable assay to screen AChE inhibitors, which is promising for the preliminary evaluation of a large number of potential candidates.

Peroxynitrite-Promoted Persulfide Prodrugs with Protective Potential against Paracetamol Poisoning.

The newly emerging persulfide prodrugs provide additional options for the profound study of persulfide, a fascinating molecule expected to intervene in biological functions and even diseases. Peroxynitrite is often the culprit in pathological processes characterized by oxidative stress, while the persulfide prodrug responsive to it is still pending. To enrich the family of redox-activated prodrugs, we designed prodrugs with a 2-oxo-2-phenylacetamide trigger, which achieved the release of persulfide via 1, 6-N, S-relay. The degradation of prodrugs and the formation of persulfides were confirmed to be peroxynitrite-responsible by the qualitative and quantitative studies based on LC-MS/MS methods and a spectrophotometry-based tag-switch strategy. Furthermore, these prodrugs showed potent peroxynitrite scavenging activity, cellular therapeutic potential against paracetamol poisoning in HepG2 and oxidative stress in H9c2, as well as desirable in vitro metabolic properties.

A Photocleavable Amphiphilic Prodrug Self-Assembled Nanoparticles with Effective Anticancer Activity In Vitro.

Accelerating degradation of prodrug is an effective strategy for improving the pharmacological action. A photocleavable amphiphilic prodrug of methotrexate-coumarin derivative-PEG conjugates (MTX-AMC-PEG) with photo-triggered breakage to release clinical drug under laser irradiation was fabricated and self-assembled into nanoparticles for chemotherapy. The nanoparticles exhibited good intracellular uptake and excellent photolysis release of MTX, which resulted in efficient anticancer activity in vitro with laser irradiation. This research provides a way to fabricate photocleavable prodrug nanoparticles with stimuli-triggered drug release behavior.

A comparative study of the antitumor efficacy of peptide-doxorubicin conjugates with different linkers

The peptide-drug conjugates caused much attention currently. The purpose of present study was to elucidate the possible synergistic effect between ligand peptide and stimuli-responsive linkage in amphiphilic peptide-drug conjugates (APDCs) with different linkers. Especially, the superiority of each strategy as well as the synergistic effect between them was carefully investigated via the parallel comparisons of the three systems throughout of the whole study. Here, we synthesized three APDCs, namely, cRGD-SS-DOX (RSSDOX), cRGD-S-DOX (RSDOX) and cRGD-VC-DOX (RVCDOX), using doxorubicin (DOX) as a model cytotoxic agent, cRGDfC as a homing peptide, and reduction cleavable disulfide (SS), noncleavable single thioether (S) or cathepsin B cleavable valine-citrulline dipeptide (VC) as linker. The APDCs showed high drug loading capacity and they were evaluated in vitro in the integrin αvβ3-overexpressing B16 cells and in vivo in tumor-bearing C57BL/6 mice. Endocytosis mechanism assay demonstrated that three types of APDCs internalized into cells through adynamin and actin depolymerizing-mediated pathway following receptor-mediated endocytosis. Notably, RSDOX or RVCDOX induced stronger antitumor efficacy, which depended on their cellular uptake levels, intracellular trafficking and the colocalization rates with lysosomes. The in vivo efficacy of RSDOX or RVCDOX was 1.4–1.7 fold of free DOX and 1.7–2.0 fold of RSSDOX, respectively. In addition, RSDOX or RVCDOX demonstrated acceptable system, tissue and blood compatibility. The compromised efficacy of RSSDOX might be due to the generation of DOX-SH during degradation of prodrug, but not DOX. Taken together, our studies suggest that certain type of APDCs can significantly decrease the toxicity of free DOX and improve therapy outcome, which provides insight for the design of peptide-drug conjugates integrating ligand peptide and stimuli-responsive linkage.

Long-Acting Diclofenac Ester Prodrugs for Joint Injection: Kinetics, Mechanism of Degradation, and In Vitro Release From Prodrug Suspension

A prodrug approach for local and sustained diclofenac action after injection into joints based on ester prodrugs having a pH-dependent solubility is presented. Inherent ester prodrug properties influencing the duration of action include their pH-dependent solubility and charge state, as well as susceptibility to undergo esterase facilitated hydrolysis. In this study, physicochemical properties and pH rate profiles of 3 diclofenac ester prodrugs differing with respect to the spacer carbon chain length between the drug and the imidazole-based promoiety were determined and a rate equation for prodrug degradation in aqueous solution in the pH range 1-10 was derived. In the pH range 6-10, the prodrugs were subject to parallel degradation to yield diclofenac and an indolinone derivative. The prodrug degradation was found to be about 6-fold faster in 80% (vol/vol) human plasma as compared to 80% (vol/vol) human synovial fluid with 2-(1-methyl-1H-imidazol-2-yl)ethyl 2-(2-(2,6 dichlorophenyl)amino)phenylacetate being the poorest substrate toward enzymatic cleavage. The conversion and release of parent diclofenac from prodrug suspensions in vitro were studied using the rotating dialysis model. The results suggest that it is possible to alter and control dissolution and reconversion behavior of the diclofenac prodrugs, thus making the prodrug approach feasible for local and sustained diclofenac action after joint injection.

Stereoisomeric Prodrugs to Improve Corneal Absorption of Prednisolone: Synthesis and In Vitro Evaluation.

A series of stereoisomeric prodrugs have been designed to examine efficacy in generating higher corneal absorption relative to prednisolone. Prodrugs have been studied and identified with LC/MS/MS and NMR analyses. Prodrugs have been characterized for aqueous solubility, buffer stability, and cytotoxicity. Cellular uptake and permeability studies have been conducted across MDCK-MDR1 cells to determine prodrug affinity towards P-glycoprotein (P-gp) and peptide transporters. Enzyme-mediated degradation of prodrugs has been determined using Statens Seruminstitut rabbit cornea (SIRC) cell homogenates. Prodrugs exhibited higher aqueous solubility relative to prednisolone. Prodrugs circumvented P-gp-mediated cellular efflux and were recognized by peptide transporters. Prodrugs (DP, DDP) produced with D-isomers (D-valine) were significantly stable against both chemical and enzymatic hydrolyses. The order of degradation rate constants observed in chemical and enzymatic hydrolyses were in the same order, i.e., L-valine-L-valine-prednisolone (LLP) > L-valine-D-valine-prednisolone (LDP) > D-valine-L-valine-prednisolone (DLP) > D-valine-D-valine-prednisolone (DDP). Results obtained from this study clearly suggest that stereoisomeric prodrug approach is an effective strategy to overcome P-gp-mediated efflux and improve transcorneal permeability of prednisolone following topical administration.

Spectroscopic characterization of both aqueous and solid-state diacerhein/hydroxypropyl-β-cyclodextrin inclusion complexes

Diacerhein, a poorly water soluble antirheumatic prodrug, was spectroscopically characterized to form inclusion complexes with hydroxypropyl-β-cyclodextrin (HPβCD) in both aqueous solution and in solid phase. Complexation with the hydrophilic carriers was used to improve the solubility and dissolution rate of the compound. The kinetics of the prodrug degradation to the active rhein in aqueous buffer solution were also investigated as a function of HPβCD concentration. The solid complexes prepared by different methods such as physical mixture, kneading, co-evaporation method and freeze dried method in 1:1M ratio, were characterized by DSC and FTIR. The dissolution profiles of solid complexes were determined and compared with diacerhein alone and their physical mixture, in the simulated intestinal fluid at 37°C. The accurate molecular spectroscopic characterization of diacerhein in the presence of different amounts of aqueous cyclodextrins was essential to determine the correct binding constants for the diacerhein/HPβCD system. The binding constants were also validated by UV spectrometry and HPLC procedure in order to compare the values from the different methods. Higuchi–Connors phase solubility method has proved not suitable when either the free or/and the complexed prodrug degrade in aqueous solution.

Biodegradable Microspheres Loaded with an Anti-Parkinson Prodrug: An in Vivo Pharmacokinetic Study

During chronic treatment with L-dopa (LD), Parkinsonian patients often experience uncontrolled motor complications due to fluctuations of the plasmatic levels of LD that result in pulsatile dopaminergic stimulation. To overcome these plasmatic fluctuations, a novel prodrug of LD, L-dopa-α-lipoic acid (LD-LA), has been proposed as a tool for achieving continuous dopaminergic stimulation. Due to slower susceptibility toward enzymatic conversion by LD-degrading enzymes (such as catechol-O-methyltransferase and monoamine oxidase), the plasma half-life of this prodrug is longer than that of LD. Moreover, the higher lipophilicity of LD-LA over LD promotes its delivery to the CNS, where the resulting levels of dopamine (DA) are kept high for a longer time than after equimolar administration of LD. To further reduce fluctuations in plasma levels of LD, LD-LA has been entrapped into biodegradable polymeric microspheres to be used as a depot system with the aim to prevent prodrug degradation and to obtain a sustained release of the intact compound. In the present work, a formulation of LD-LA loaded microspheres (characterized for drug loading, size, morphology, thermal properties, and in vitro prodrug release) has been administered subcutaneously to rats, and the resulting levels of LD and DA in plasma and striatal tissue, respectively, have been monitored. A good correlation between the in vitro release kinetics and the time range during which the formulation alters the LD/DA tissue levels in vivo was observed, suggesting that the polymeric microsphere matrix protects the loaded prodrug from chemical and enzymatic degradation and controls its release. Interestingly, LD-LA microspheres provided sustained levels of DA neurotransmitter in the striatum nucleus for up to 4 days after a single administration. In conclusion, a polymeric microsphere formulation of LD-LA is an attractive medicine for treating Parkinson's disease (PD) symptoms, avoiding motor complications.

Kinetic assessment of luminal degradation of orally effective prodrugs for rational drug development

Although prodrugging (prodrug derivatization) is a powerful technique for improving the pharmacokinetic characteristics of drugs, the intestinal pharmacokinetics of prodrugs has yet to be elucidated fully. A previous article reported the kinetic requirement of prodrugs to overcome membrane barriers. In the present article, the luminal degradation of prodrugs was kinetically assessed to understand crucial factors in the intestinal absorption of prodrugs and to show a rational development procedure. A kinetic model equation involving luminal degradation clearance (CLdeg) was derived, and CLdeg was estimated according to the equation with in vitro and in vivo reported data of two kinds of ampicillin prodrugs (lenampicillin and pivampicillin) and one acyclovir prodrug (valacyclovir). For lenampicillin ((2,2-dimethyl-1-oxopropoxy)methyl ester derivative), CLdeg was approximately 1.7 times as large as absorption clearance (CLabs), whereas for pivampicillin ((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl ester derivative), CLdeg was approximately one tenth of CLabs. For valacyclovir (acyclovir prodrug), CLdeg was negligible. These results indicate that not only membrane permeability but also luminal stability should be assessed for the rational development of orally effective prodrugs, and that luminal stabilization can improve the intestinal absorption of prodrugs. A procedure was proposed to develop orally effective prodrugs considered for luminal degradation as well as membrane permeability. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1078–1086, 2010

Self-assembled drug delivery systems. Part 5: Self-assemblies of a bolaamphiphilic prodrug containing dual zidovudine

A bolaamphiphilic prodrug containing dual zidovudine, pentadecanedioyl dizidovudine (PDDZ), was prepared. The vesicular self-assemblies were formed in aqueous media through injecting the methanol solution of PDDZ into water. Hydrophobic interaction between lipid chains should drive molecular self-assembly. The nonionic surfactant, Tween 20, was used to increase the physical stability of self-assemblies because the surfactant micelles could prevent the assemblies from aggregating. The doping hydroxylpropylmethylcellulose (HPMC) slowed down the degradation of prodrugs due to adsorption. The self-assemblies were nanoscale with the mean particle size of 156 nm. Degradation of PDDZ was very slow in buffered solutions, but very rapid in enzyme and plasma, and the parent drug zidovudine (AZT) was the unique product. PDDZ self-assemblies showed strong anti-HIV activity on MT4 cell model. The 50% effective concentration (EC 50) of PDDZ was 5 nM, equal to that of AZT. PDDZ was rapidly eliminated from circulation and mainly distributed into liver, spleen and testis followed by the rapid production of AZT after intravenous administration of the self-assemblies to rabbits. Macrophages in liver, spleen and testis are the reservoir of HIV so that the macrophage targeting effect of PDDZ self-assemblies would benefit to anti-HIV therapy. The self-assemblies composed of bolaamphiphilic PDDZ are a promising self-assembled drug delivery system (SADDS).

Absorption of ester prodrugs in Caco-2 and rat intestine models.

The aim of this study was to elucidate the absorption mechanism in Caco-2 and rat intestine models in order to improve the accuracy of prediction of oral absorption of ester prodrugs. Pivampicillin and cefcapene pivoxil hydrochloride (CFPN-PI), ester-type oral antibiotics, were chosen as model ester prodrugs. The level of esterase activity in Caco-2 cells was lower than that measured in the rat jejunum when p-nitrophenyl acetate was used as a substrate. Almost complete ester hydrolysis occurred before the ester prodrugs reached the basolateral side of the monolayer, and the disappearance of prodrugs was thought to be due to metabolism or transport after addition to the apical side of the monolayer. When pivampicillin and CFPN-PI were used, the amounts of ampicillin and cefcapene (CFPN) produced by hydrolysis of prodrugs were increased because intracellular degradation of prodrugs resulted in intracellular accumulation. On the other hand, when ampicillin or CFPN was used, only a small amount of the drug reached the basolateral side of the monolayers and no intracellular accumulation was observed. The permeability of CFPN-PI, the solubility of which is dependent on the acidity of gastric juice, across a Caco-2 monolayer or rat intestine, was also investigated by using an in vitro system that mimics the physiological state of the human gastrointestinal tract. The oral absorption of CFPN-PI in humans is predicted to be good either in the Caco-2 model or in the rat intestine model. It is concluded that our system may be a valuable tool for evaluation of oral absorption of ester prodrugs metabolized during permeation through the intestinal epithelium. Broader evaluation of such a system is warranted.

Degradation of camptothecin-20( S)-glycinate ester prodrug under physiological conditions

We have compared the strikingly different decomposition pathways for camptothecin-20( S)-acetate -acetate and camptothecin-20( S)-glycinate in phosphate buffered saline, human plasma and blood. The aliphatic ester analog camptothecin-20( S)-acetate demonstrated excellent stability in the above fluids for many hours with minimal hydrolysis, while the camptothecin-20( S)-glycinate analog (differing solely by the presence of an amino group) underwent rapid and essentially complete decomposition. Reversed-phase high performance liquid chromatography (RP-HPLC) with electrospray ionization–mass spectral (ESI–MS) detection was then used to correlate structural information for camptothecin-20( S)-glycinate decomposition products. ESI–MS detection indicated the ring-opened carboxylate form of camptothecin and the ring-opened degradation product co-elute near the solvent front, while the latest eluting decomposition product was the closed-ring lactone form of camptothecin. A novel decomposition product with intermediate retention time displayed an identical mass-to-charge ratio as camptothecin-20( S)-glycinate ester but a strikingly different fragmentation pattern. The LC–ESI–MS evidence of a novel camptothecin prodrug degradation pathway is provided in this report.

Hydroxypropyl-beta-cyclodextrin increases the aqueous solubility and stability of pilocarpine prodrugs.

The effects of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on the aqueous solubility and stability of two lipophilic bispilocarpine prodrugs were investigated at pH 7.4. The solubility of prodrugs was studied by phase-solubility method (0-72.5 mM HP-beta-CD). The stability of one of the prodrugs was investigated as a function of temperature (40 degrees C-70 degrees C) and HP-beta-CD concentration (0-72.5 mM). The apparent rate constants (k1, k2) for degradation of prodrug in 1:1 and 1:2 inclusion complexes and apparent stability constants (K1:1, K1:2) were calculated by the curve-fitting method. The phase-solubility diagrams were classified as Ap-type and the apparent stability constants (K1:1, K1:2) for 1:1- and 1:2-inclusion complexes were calculated to be 143-815 M-1 and 29-825 M-1, respectively. The stability of prodrug increased as a function of HP-beta-CD concentration over the studied temperature range. The shelf-life (t90%, calculated by the Arrhenius equation) of the prodrug in 72.5 mM HP-beta-CD solution increased 5.1-fold and 6.1-fold at 25 degrees C and 4 degrees C, respectively. The solubility of the prodrugs was shown to increase markedly in phase-solubility studies. The degradation rate of prodrug in stability studies was shown to be slower in the 1:2-complex than in the 1:1-complex and the relative amounts of complex species were found to be dependent on CD concentration.

Biodegradation characteristics of acyclovir 2′-esters by respiratory carboxylesterases: Implications in prodrug design for intranasal and pulmonary drug delivery

Eight aliphatic and one aromatic 2′-ester prodrugs of 9-(2-hydroxyethoxymethyl)guanine (acyclovir) were incubated with rat nasal mucosal homogenate in vitro to characterize their degradation kinetics by carboxylesterase. Lineweaver-Burk plots revealed that prodrugs with longer linear carboxylic side chains possess higher V max and smaller K m values than esters with shorter side chains, probably as a result of improved lipophilicity. Under subsaturating conditions, the rate and extent of prodrug degradation assume a direct relationship with acyl side chain length. Lengthening of linear carboxylic chains from three to eight carbons resulted in a more than 600-fold increase in first-order degradation rate constants. Esters with branched acyl chains are more resistant to nasal carboxylesterase-mediated cleavage. Acyclovir benzoate, on the other hand, did not afford specific protective effect against nasal carboxylesterase, although such an effect was found in plasma. Comparison of the second-order rate constants for binding and catalysis ( V max/ K max) indicated that the catalytic affinity of nasal carboxylesterase differs significantly from that of ester hydrolase in plasma. Comparison of hexanoate degradation kinetics in tracheal, pulmonary parenchymal, and nasal homogenates suggested that nasal mucosa possesses the highest carboxylesterase activity in the respiratory tract. The uniquely rich presence of this enzyme in the nasal cavity forms a remarkable enzymatic barrier to prodrug design for systemic delivery.

P2 Regional differences in prodrug degradation in the rat

P2 Regional differences in prodrug degradation in the rat

Liposome-Incorporated 3′,5′-O-Dipalmitoyl-5 -Fluoro-2′-Deoxyuridine as a Slow-Release Anti-Tumor Drug Depot in Rat Liver Macrophages

We synthesized the 3',5'-O-dipalmitoyl derivative of 5-fluoro-6-[3H]-2'-deoxyuridine and incorporated it into the bilayers of multilamellar liposomes (400 nm diameter) of various lipid compositions. The prodrug-containing liposomes were incubated with rat liver macrophages (Kupffer cells) in monolayer culture and with lysosomal fractions from whole rat liver homogenates. The release of water-soluble radioactive degradation products from the cells was measured and we found the rate of release strongly dependent on the lipid composition of the liposomes. After 4 hours of incubation the release of radioactivity was 9-fold higher from egg-phosphatidylcholine/phosphatidylserine/cholesterol liposomes than from distearoylphosphatidylcholine/dipalmitoylphosphatidylglycerol/cholest ero l or dioctadecyl-sn-glycero-phosphorylcholine/dipalmitoylphosphatidylg lycerol/ cholesterol liposomes. A somewhat less pronounced difference in rate of prodrug degradation was found when the liposomes were incubated with lysosomal fractions. The water-soluble products that were formed showed anti-tumor activity against C26-adenocarcinoma tumor cells in vitro. Preliminary evidence suggests this activity to be caused by 5-fluoro-2'-deoxyuridine. We conclude that incubation of liposomes of varied composition containing diacylated 5-fluoro-2' deoxyuridine derivatives with Kupffer cells in culture, results in the formation of an intracellular prodrug depot in these cells from which compounds with anti-tumor activity are released with controllable rates.

Synthesis and kinetic stability studies of progesterone derivatives

The 3-potassium salt of carbomethoxyl amine derivative along with the betaine and pyridinium hydrazide chloride derivatives of progesterone were synthesized and their kinetics of decomposition in aqueous solution was studied. The rates of hydrolysis yielding progesterone was determined at 25°C over the pH range of 1–13 at an ionic strength of 0.5. The pH-rate profiles were accounted for by the specific acid-, base- and water-catalyzed spontaneous decomposition of these prodrugs. Maximum stability occurred at pH 6–7 in which region water was the principal catalyst in the decomposition pathway. Various buffer and temperature effects were also evaluated. In the slightly alkaline to basic medium the principal mode of prodrug degradation may be the cleavage of the carbon nitrogen bond by direct neucleophilic attack of the water molecule or the hydroxide ion at the 3-carbon of the A ring.

Potential improvement in shelf life using the prodrug approach. II. A systematic examination of kinetic requirements.

The utilization time (UT) for a solution of a prodrug that is rapidly and completely converted to drug in the blood may be longer than the time for 10% loss of the initial concentration. The UT for an intravenous prodrug solution is the period during which the total prodrug and drug concentration exceeds 90% of the initial concentration. The influence of the rate of prodrug degradation (knc), its conversion (kc) to drug, and the subsequent drug degradation (kh) on the UT of a stored solution was examined by simulating the prodrug and drug concentration-time courses. The ratio of the shelf life of a prodrug solution to that of the parent drug (UTratio) was calculated using a wide range of values for the three rate constants. Three-dimensional plots relating the UTratio to the kc, knc, and kh values provide a basis for making a priori assessments of kinetic requirements for designing a prodrug to increase storage time. A parenteral prodrug intended to increase storage time may have a larger overall rate of loss than the parent drug, but it must have a smaller degradation rate (knc less than kh) to be successful. The UT for an oral prodrug solution depends upon the bioavailability of the prodrug relative to the drug in addition to the values for knc, kc, and kh. Two ampicillin prodrugs were used as models to calculate actual UTratio versus pH profiles. Intravenous solutions showed modest gains in the UTratio in the acid region, whereas oral solutions reached a UTratio as high as 22 by combining favorable rate constants with increased bioavailability.(ABSTRACT TRUNCATED AT 250 WORDS)