A validated LC-MS/MS method for determination of antiviral prodrug molnupiravir in human plasma and its application for a pharmacokinetic modeling study in healthy Egyptian volunteers.

A fully validated, simple, rapid and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine NHC (N-hydroxycytidine), the active metabolite of Molnupiravir (MOL) in human plasma; one of the limited treatment options for SARS-CoV-2 in plasma of healthy volunteers. The internal standard (IS) used was ribavirin. The extraction of analyte and IS from plasma was performed using acetonitrile as a solvent for protein precipitation. Agilent Zorbax Eclipse plus C18, 4.6 × 150 mm, (5 µm) was used for chromatographic separation using a mixture of methanol0.2 % acetic acid (5:95, v/v) as a mobile phase that was pumped at a flow rate of 0.9 mL/min. Detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing positive ESI interface using API4500 triple quadrupole tandem mass spectrometer system, with the transitions set at m/z 260.10 â†’ 128.10 and 245.10 â†’ 113.20 for NHC and IS respectively. Method validation was performed in accordance with United States FDA bioanalytical guidance. The concentration range of 20.0-10000.0 ng/mL was used to establish linearity via weighted linear regression approach (1/x2). Moreover, the analyzed pharmacokinetic data from twelve Egyptian healthy volunteers were used to develop a population pharmacokinetic model for NHC. The developed model was used to perform simulations and evaluate the current MOL dosing recommendations through calculating the maximum concentration (Cmax) "the safety metric" and area under the curve (AUC0-12 h) "the efficacy metric" for 1000 virtual subjects. Geometric mean ratios (GMR) with their associated 90% confidence intervals (CI) compared to literature values were computed. Geometric means of simulation-based Cmax and AUC0-12 were 3827 ng/mL (GMR = 1.05; 90% CI = 0.96-1.15) and 9320 ng.h/mL (GMR = 1.04; 90% CI = 0.97-1.11), respectively indicating that current MOL dosage can achieve the therapeutic targets and dose adjustment may not be required for the Egyptian population. The developed model could be used in the future to refine MOL dosage once further therapeutic targets are identified.

Simultaneous analysis by LC-MS/MS of 22 ketosteroids with hydroxylamine derivatization and underivatized estradiol from human plasma, serum and prostate tissue.

This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 µl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11ß-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11ß-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids.

Development and validation of an LC-MS/MS method for pharmacokinetic study of methoxyamine in phase I clinical trial.

Methoxyamine (MX) is the first DNA base-excision-repair (BER) inhibitor evaluated in humans. This work described the development and validation of an LC-MS/MS method for quantitative determination of MX in human plasma. In this method, MX and its stable isotope methoxyl-d(3)-amine (MX-d3 as internal standard) were directly derivatized in human plasma with 4-(N,N-diethylamino)benzaldehyde. The derivatized MX and IS were extracted by methyl-tert-butyl ether, and separated isocratically on a Xterra C18 column (2.1 mm × 100 mm) using an aqueous mobile phase containing 45% acetonitrile and 0.4% formic acid at a flow rate of 0.200 ml/min. Quantitation of MX was carried out by multiple-reaction-monitoring (MRM) mode of positive turbo-ion-spray tandem mass spectrometry. This method has been validated according to FDA guidelines for bioanalytical method. The linear calibration range for MX was 1.25-500 ng/ml in human plasma with a correlation coefficient≥0.9993. The intra- and inter-assay precision (%CV) at three concentration levels (3.50, 45.0 and 450 ng/ml) ranged 0.9-1% and 0.8-3%, respectively. The stability studies showed that MX met the acceptable criteria under all tested conditions. The method developed had been applied to the determination of plasma MX concentrations in the first-in-human phase I clinical trial, and PK data were presented.

Hemolytic potential of structurally related aniline halogenated hydroxylamines.

This study was undertaken to investigate the hemolytic potential of several structurally related aniline halogenated phenylhydroxylamines based on their decreasing electro negativity. The compounds compared are phenylhydroxylamine (PHA) and para-fluoro-, para-bromo-, and para-iodo-phenylhydroxylamines. Red blood cells of male Sprague-Dawley rats were labeled with radioactive chromium-51 and exposed to the test agent before being infused into the tail vein of isologous rats. The time course of blood radioactivity was monitored. The stability of some selected halogenated aniline analogs was also determined in blood. All four tested hydroxylamines produced dose-dependent reduction in the circulating labeled red blood cells indicating their destruction and loss. The most pronounced reduction was observed at doses from 175 to 250 microM. The dose of 100 microM appeared to be the threshold limit. The para-iodo-PHA was two times more toxic than para-fluoro-PHA in the destruction of red blood cells in rats.

Measurement of anti-cancer agent methoxyamine in plasma by tandem mass spectrometry with on-line sample extraction.

In this work, we present the development and validation of a tandem mass spectrometry method for the quantitative determination of methoxyamine (CH3ONH2), a potential new chemotherapeutic agent, in human and mouse plasma. Methoxyamine together with the internal standard (I.S.) methoxyl-D3-amine was directly derivatized in plasma sample with a novel chemical agent 4-(N,N-diethylamino)benzaldehyde. The product solution was injected into an on-line Oasis HLB extraction column (2.1 mm x 20 mm) for analyte extraction. After the elution of extractives, the derivatized analytes were monitored by the positive-electrospray-ionization mass spectrometry (ESI-MS-MS). The structures of derivatized analytes were elucidated by fragmentation. Quantitation of plasma methoxyamine was carried out by the multiple reaction monitoring (MRM) mode. This method had a linear calibration range of 1.00-1000 ng/ml with a correlation coefficient of 0.9999 for methoxyamine in both human and mouse plasma. The limit of detection (LOD) and limit of quantification (LOQ) for methoxyamine in plasma were 0.150 and 0.500 ng/ml, respectively. It was demonstrated that the method had high recovery and accuracy (90.1-94.7 and 90.1-96.3%), as well as excellent intra- and inter-assay precision (2.2 and 3.7%), at three concentration levels (5.00, 50.0, 500 ng/ml). This method has been used to analyze the plasma levels of methoxyamine in samples obtained from male CD1 mice after bolus intraperitoneal injection of 2, 5 and 20mg methoxyamine hydrochloride (CH3ONH2.HCl) per kilogram mouse.

New strategy for antedrug application: development of metalloproteinase inhibitors as antipsoriatic drugs.

Phosphonamide-based inhibitors were synthesized and evaluated for the inhibitory activities against the shedding of epidermal growth factors, amphiregulin and heparin-binding EGF-like growth factor, that would participate in the development of psoriasis. All compounds exhibited excellent inhibitory activities for these EGF sheddings; however, they also inhibited matrix metalloproteinases (MMPs). To avoid adverse effects reported by the clinical development of MMP inhibitors, the antedrug concept was introduced. Among the phosphonamide inhibitors, the 2,2,2-trifluoroethyl ester 8d and 2,2-difluoroethyl ester 8c showed rapid decomposition in human plasma, which is an essential property for the antedrug. Topical applications of these compounds significantly suppressed TPA-induced epidermal hyperplasia in murin skin, a model of psoriasis. These results suggested that the phosphonamide-based inhibitors have a therapeutic potential for the treatment of psoriasis as an antedrug application.

[Normal values of plasma methoxyamines in the setting of renal insufficiency and peri-operative stress. Consequences for the etiological diagnosis of hypertension]. / Valeurs normales des méthoxyamines plasmatiques en situation d'insuffisance rénale et de stress péri-opératoire.

INTRODUCTION: HPLC plasma methoxyamines measurements are the updated technique for the diagnosis of adrenergic hypersecretion. Their reliability meets that of urinary measurements. Significance of increased values is not yet fully established for the etiological diagnosis of hypertension in some situations, especially in case of renal insufficiency and in the peri-operative period. The aim of this study is to define the "normal" range of the values of plasma methoxyamines in both of those conditions. PATIENTS AND METHODS: in a General and Endocrine Surgical Unit, 3 homogeneous group of 20 patients each have been studied: group 1, control (patients awaiting thyroidectomy); group 2, patients on maintenance hemodialysis submitted for hyperparathyroidism; group 3, patients submitted to digestive surgery. Measurements were done pre-operatively in group 1, pre and post-operatively in group 2, and post-operatively in group 3. RESULTS: in comparison to the control (11.8 nmol/l), we observed in group 2 a 18 fold increase preoperatively, and a 29 fold increase at post-operative day 1. In group 3, we observed a 2.3, 2.7 and 2 fold increase at post-operative days 1,2 and 3 respectively. All those results were statistically significant. CONCLUSION: Results of measurements of plama methoxyamines should always be matched to the serum creatinine levels. They are meaningful for the diagnosis of endocrine origin of hypertension only late after the early post-operative period.

[The biological diagnosis of pheochromocytoma]. / La stratégie du diagnostic biologique du phéochromocytome.

Pheochromocytomas are tumors secreting large amount of catecholamines. Elevation of blood pressure is the classical manifestation but frequently the tumors are silent and they have to be screened systematically. The biological diagnosis is essential to affirm the tumor before any imaging procedure. It needs to select the most sensitive and specific methods. The sensitivity of VMA, urinary catecholamines and plasma catecholamines assays is respectively 70%, 75%, 85%. Determination of methoxyamines in the urine or better in the plasma reaches a sensitivities of 98%. This represents the best tool for the diagnosis of pheochromocytomas. Only renal or heart failure decrease the specificity of the plasma methoxyamines assay.

[Biological diagnosis of pheochromocytoma: impact of technological improvement]. / Le diagnostic biologique du phéochromocytome: l'impact de l'évolution technologique.

Laboratory diagnosis of pheochromocytoma must give evidence of increased catecholamine production. This requires measurement of catecholamines and their metabolites (normetanephrine NMN, metanephrine MN and/or VMA) in urine or in plasma. The various assays can be also performed during dynamic test that stimulate or inhibit catecholamine release. The recent introduction in biochemistry of high performance liquid chromatography coupled to electrochemical detection (HPLC-ED) has greatly reduced drug-induced interference and has therefore narrowed the reference value range. The two groups of compounds that have most benefited from such analytical improvements are urinary metanephrines and VMA. The technical progress has greatly simplified the laboratory diagnosis of pheochromocytoma both by improving the reliability of already available compounds and by favouring the development of news markers. However, the diagnostic sensitivity of the various urinary and plasmatic markers remains very unequal and the diagnosis of pheochromocytoma requires a carefully planned sequence of studies including appropriate biochemical tests able to affirm or to exclude the diagnosis with a high degree of security while reducing the duration and cost of the investigation. Among urinary markers, metanephrines remain the most direct indices of catecholamine hypersecretion and provide the most reliable biochemical indicators of the existence of pheochromocytoma. The diagnostic sensitivity of urinary metanephrines (about 98%) greatly exceeds that of catecholamines and VMA (60-70%). These differences are related to the diversity and specificity of physiological mechanisms involved in the synthesis, the release and inactivation of markers (catecholamines, metanephrines, VMA) and to the variety of clinical presentations and secretory patterns of pheochromocytomas. Considering the practical necessity of simplifying the collection of laboratory samples, use of plasma assays for the diagnosis of pheochromocytoma has become increasingly routine. However, plasma catecholamines--even when assayed during the clonidine suppression test--have not fully lived up to expectations. The diagnostic sensitivity is far better (about 98%) with the recently developed assays of plasma methoxyamines which, owing to their long half-life, provide long-lasting indicators of the catecholamine discharge and are elevated even in tumors without clinical expression. Laboratory diagnosis is relatively easy when the patient bears a large tumor releasing considerable amounts of catecholamines and metabolites; it becomes more challenging in the case of small tumors or of pretumoral hyperplasia in which only the most reliable biochemical markers are able to confirm the diagnosis of pheochromocytoma.

Aniline-, phenylhydroxylamine-, nitrosobenzene-, and nitrobenzene-induced hemoglobin thiyl free radical formation in vivo and in vitro.

We have employed the ESR spin trapping technique in vivo to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats following administration of either aniline, phenylhydroxylamine, nitrosobenzene, or nitrobenzene. This DMPO adduct was a six-line, strongly immobilized, radical adduct. Using rat red blood cells, both phenylhydroxylamine and nitrosobenzene were able to induce the formation of the DMPO/glutathiyl free radical adduct and the same DMPO/hemoglobin thiyl free radical adduct was detected in in vivo samples. In experiments using purified rat oxyhemoglobin, a four-line, weakly immobilized, DMPO/hemoglobin thiyl free radical adduct was detected, in addition to the six-line strongly immobilized adduct. When this study was repeated using human red blood cells, we detected only the DMPO/glutathiyl free radical adduct and, when purified human oxyhemoglobin was employed, only the four-line, weakly immobilized, DMPO/hemoglobin thiyl radical adduct could be detected. In a study using reduced glutathione, we found that phenylhydronitroxide free radicals were reduced by glutathione and that glutathione was concomitantly oxidized to its thiyl free radical. We propose that the species responsible for the oxidation of the thiols to yield the thiyl free radicals in vivo and in vitro was the phenylhydronitroxide radical produced from the reaction of phenylhydroxylamine with oxyhemoglobin.

Detoxication of N-oxygenated arylamines in erythrocytes. An overview.

1. Reactive N-oxygenated arylamines, namely, N-hydroxyarylamines and nitrosoarenes, are toxic, mutagenic, carcinogenic and allergenic. 2. Erythrocytes are a very sensitive target for these compounds, but little is known of the detoxication capacity of these cells. 3. This overview considers the most important reactions of p-substituted N-oxygenated arylamines in red cells, namely, (i) ferrihaemoglobin formation by N-hydroxyarylamines with concomitant co-oxidation to nitrosoarenes; (ii) compartmentation of nitrosobenzenes by ligation to deoxyhaemoglobin, (iii) reactions of nitrosobenzene with glutathione, (iv) adduct formation of nitrosobenzenes with thiol groups of haemoglobin. 4. To predict the metabolic fate of N-oxygenated arylamines in red cells, the respective kinetic parameters of reactions (i) to (iv) have been determined, and indicate good linear free energy correlations (pH 7.4, 37 degrees C). These data may help to estimate the detoxication capacity of erythrocytes in vivo.

Biologic activity of hydroxylamine: a review.

Although hydroxylamine, as such, is a product of normal cellular metabolism it is also a potent mutagen in vitro. However, in spite of this potential, it has not been shown to possess carcinogenic capabilities. Indeed, this chemical has demonstrated carcinostatic activity against certain tumors in animals. In addition, hydroxylamine has been shown to inactivate or inhibit a number of cellular enzymes and some viruses in vitro. It is also a skin irritant and sensitizer. It causes dermatitis and it is corrosive to the eyes. Acute and chronic exposures to hydroxylamine have caused methemoglobinemia and sulfhemoglobinemia.

Hemolysis induced by 6-N-hydroxylaminopurine riboside, an adenosine analogue.

6-N-Hydroxylaminopurine riboside (HAPR) was studied in man because of its therapeutic activity in several transplanted animal neoplasms. It was not cross-resistant to other antimetabolites useful in the treatment of human neoplasia. HAPR produced marked hemolytic anemia at doses far below those that might have produced any cytotoxic or therapeutic effect. There was evidence of hemolysis at total doses as low as 0.5 mg/kg given intravenously. For man, HAPR is one of the most active hemolytic drugs.