Michael Brandl

Physicochemical properties of the nucleoside prodrug R1626 leading to high oral bioavailability.

The nucleoside analog R1479 is a potent and highly selective inhibitor of NS5b-directed hepatitis C virus (HCV) RNA polymerase in vitro. Because of its limited permeability, lipophilic prodrugs of R1479 were screened. Selection of the prodrug involved optimization of solubility, permeability, and stability parameters. R1626 has dissociation constant, intrinsic solubility, log partition coefficient (n-octanol water), and Caco-2 permeability of 3.62, 0.19 mg/mL, 2.45, and 14.95 × 10−6 cm/s, respectively. The hydrolysis of the prodrug is significantly faster in the Caco-2 experiments than in hydrolytic experiments, suggesting that the hydrolysis is catalyzed by enzymes in the cellular membrane. Using GastroPlus™, the physical properties of R1626 successfully predict the dose dependence of the pharmacokinetics in humans previously studied. The program predicts that if the particle size of R1626 is less than 25 μm, it will be well absorbed. Prodrugs with a solubility of greater than 100 μg/mL and permeability in the Caco-2 assay greater than 3 × 10−6 cm/s are expected to achieve a high fraction absorbed.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 2: Synthesis and structure―activity relationships of benzothiazine-substituted quinolinediones

A new series of benzothiazine-substituted quinolinediones were evaluated as inhibitors of HCV polymerase NS5B. SAR studies on this series revealed a methyl sulfonamide group as a high affinity feature. Analogues with this group showed submicromolar potencies in the HCV cell based replicon assay. Pharmacokinetic and toxicology studies were also performed on a selected compound (34) to evaluate in vivo properties of this new class of inhibitors of HCV NS5B polymerase.

Influence of Ferrous Sulfate on the Solubility, Partition Coefficient, and Stability of Mycophenolic Acid and the Ester Mycophenolate Mofetil

ABSTRACT Studies were performed to (1) evaluate whether the presence of iron affected the physicochemical properties of mycophenolate mofetil (MMF) and mycophenolic acid (MPA), and (2) determine whether alteration of these properties was indicative of formation of an MMF–iron complex. The solubility, stability (chemical reactivity), and partitioning properties of MMF and MPA were evaluated over a pH range of 2–7 in the presence and absence of ferrous sulfate. In addition, the solubility and partitioning properties of MMF were assessed after the MMF drug product, CellCept® capsules, was combined with an iron tablet (Fero-Gradumet®, ferrous sulfate, tablets). The results of studies showed that: The solubility of MMF in the presence of ferrous sulfate was generally unaffected over a pH range of 2–7; a small increase in solubility was observed in pH 5.2 buffer solution. The solubility of MPA decreased in pH 5.2 and 7.0 buffer solutions. Both MMF and MPA were more stable in the presence of ferrous sulfate at pH 2.0; ferrous sulfate had no effect on the stability of MMF and MPA at pH 7.0. Overall, the partitioning of MMF and MPA was unaffected by the addition of ferrous sulfate. The solubility and partitioning of MMF from CellCept® capsules combined with Fero-Gradumet® (ferrous sulfate) tablets showed a twofold increase in aqueous solubility of MMF as well as increased concentration of MMF in both the n-octanol and aqueous phases, leading to a decrease in the octanol/water partition coefficient due to a reduction in pH of the aqueous phase. Based on these results, it was concluded that the physicochemical properties of MMF and MPA were generally not affected by the presence of ferrous sulfate. Further, the presence of ferrous sulfate did not suggest the formation of an MMF–iron complex.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 4: structure-based design, synthesis, and biological evaluation of benzo[d]isothiazole-1,1-dioxides

A series of benzo[d]isothiazole-1,1-dioxides were designed and evaluated as inhibitors of HCV polymerase NS5B. Structure-based design led to the incorporation of a high affinity methyl sulfonamide group. Structure-activity relationship (SAR) studies of this series revealed analogues with submicromolar potencies in the HCV replicon assay and moderate pharmacokinetic properties. SAR studies combined with structure based drug design focused on the sulfonamide region led to a novel and potent cyclic analogue.

Reactivity of Valganciclovir in Aqueous Solution

The rates of hydrolysis of valganciclovir to ganciclovir and L-valine and isomerization of the R and S diastereomers of valganciclovir in aqueous buffer solution from pH 3.8 to 11.5 were determined at 37°C. The kinetics of hydrolysis were first order for at least two half-lives in neutral and basic solutions. In acidic solutions where less than 10% degradation occurred, the rate of hydrolysis was determined assuming a first-order loss in drug. At 37°C and pH 7.08, the half life is 11 h. The maximum stability at the pH values studied occurred at pH 3.81 with a half life of 220 days. The kinetics of the approach to equilibrium for the isomerization were first order and the ratio of the R:S isomer at equilibrium was 52:48. Isomerization was approximately 10 fold faster than hydrolysis over the pH range studied with a half-life at pH 7.01 of 1 h. The maximum stability toward isomerization (t1/2 > 533 h) occurs at a pH below 3.8. The pH-rate profile for the hydrolysis and the isomerization reaction are best described by hydroxide ion catalyzed mechanisms. In acidic and neutral solutions, the hydroxide reacts with the protonated form of the drug, while in basic solutions, the hydroxide reacts with the neutral form of the drug.

Degradation of tobramycin in aqueous solution

AbstractThe kinetics of degradation of tobramycin (Ne-De-Ka) in aqueous solution was studied as a function of pH. Tobramycin hydrolyzes in acidic solution to yield kanosamine (Ka-OH) and nebramine (Ne-De-OH) with a pseudo first-order rate constant of 2.7 × 10−6 s−1 in 1 N HCl at 80°C. The activation energy for the acid catalyzed hydrolysis is 32 kcal mol−1. In basic solution, the hydrolysis products are deoxystreptamine (De-OH), nebramine (Ne-De-OH) and deoxystreptamine-kanosaminide (HO-De-Ka). The pseudo first-order rate constant for the hydrolysis in 1 N KOH is 1 × 10−8 s−1 at 80°C. The activation energy for the base catalyzed hydrolysis is 15 kcal mol−1. Tobramycin is very stable towards hydrolysis at neutral pH; however, it rapidly oxidizes giving several products including De-OH, Ne-De-OH, and HO-De-Ka. In pH 7 phosphate buffer (0.01 M), the t90 value is 70 hr at 80°C.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 3: synthesis and optimization studies of benzothiazine-substituted tetramic acids.

Benzothiazine-substituted tetramic acids were discovered as highly potent non-nucleoside inhibitors of HCV NS5B polymerase. X-ray crystallography studies confirmed the binding mode of these inhibitors with HCV NS5B polymerase. Rational optimization of time dependent inactivation of CYP 3A4 and clearance was accomplished by incorporation of electron-withdrawing groups to the benzothiazine core.

Isolation and identification of ester impurities in RG7128, an HCV polymerase inhibitor

RG7128 is a di-ester prodrug of a cytidine analog for the treatment of hepatitis C virus (HCV) infection. The structures of nine low level impurities (0.05-0.10%) in RG7128 drug substance were elucidated. The majority of the impurities were formed during the synthesis of the prodrug from the parent drug. Structural elucidations of the impurities were achieved either by enrichment of the impurities using preparative chromatography followed by spectroscopic techniques or by confirmation with a reference sample. Heart-cut and recycle chromatographic techniques were applied to purify closely eluting isomers of RG7128.

Chemical stability of 4'-azidocytidine and its prodrug balapiravir

Background: R1479, a 4′-azidocytidine nucleoside analog, was developed for the treatment of Hepatitis C virus infection. Balapiravir (R1626) is the tri-isobutyrate ester prodrug of R1479 under clinical development to improve exposure of R1479 upon oral administration. Objective: The chemical stability and the rate of azide release of R1479 and balapiravir were studied. Methods: R1479 and balapiravir solutions were prepared at different pH values and stored at various temperatures. An ion pair high-performance liquid chromatography (HPLC) method with gradient elution was employed to analyze the prodrug, parent, and degradation products. Azide was measured using a reversed phase HPLC method with UV detection after formation of the 3,5-dinitrobenzoyl azide derivative with 3,5-dinitrobenzoyl chloride. The data were analyzed using initial rate and conventional first-order kinetic methods. Results: R1479 degrades to cytosine and azide in aqueous solutions, whereas balapiravir mainly degrades to R1479 and mono- and diesters of R1479. The rates of azide release from R1479 and balapiravir were generally comparable with the corresponding amount formed of cytosine. Conclusion: Azide release is pH dependent and is faster in acidic solutions than in neutral solutions. The amount of azide released is significantly less from balapiravir than that from R1479, suggesting a potential advantage of the prodrug over the parent drug.

Degradation Products of Mycophenolate Mofetil in Aqueous Solution

The thermal and peroxide-catalyzed degradation products of mycophenolate mofetil (1) were studied in aqueous solution at pH 2.0, 3.5, 6.0, and 8.2. The major thermal degradation product observed was mycophenolic acid (2). At pH 6.0 and 8.2, 2 was the only product observed in the absence of peroxide, while at pH 2.0 and 3.5, the lactone analogue of mycophenolic acid (5), a hydroxylactone due to oxygenation of the double bond (6), and an unidentified product were formed. Compound 6 degraded to 4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-car baldehyde (9) on prolonged storage and was present in the sample stressed at pH 2. Mycophenolic acid (2), the N-oxide of mycophenolate mofetil (3), the hydroxylactone of mycophenolic acid (6), and the erythro form of 4-methoxy-5-methyl- 2-(2-methyl-5-oxo-tetrahydro-furan-2-yl)-3,6-dihydro-2H-1, 7-dioxa-as-indacen-8-one (8) were observed in the presence of hydrogen peroxide at pH 3.5, 6.0, and 8.2. In addition, at pH 8.2, 4-hydroxy-4-(4-methoxy-5-methyl-8-oxo-2,3,6, 8-tetrahydro-1,7-dioxa-as-indacen-2-yl)-pentanoic acid (7) was seen. Peroxide-stressed samples at pH 2.0 gave no major degradation peaks, but a small amount of the hydroxylactone of mycophenolic acid (6) was formed.