Parameters determining isotretinoin teratogenicity in rat embryo culture

At the in vitro threshold serum concentration of 500 ng/ml, isotretinoin induces defects of visceral arch development in 9.5-day rat embryos grown in culture for 48 h. Experiments were performed to determine the minimum period of exposure necessary to induce these arch defects and whether an increase in concentration of isotretinoin could compensate for reduced exposure time. The results showed that a minimum 6-h exposure to 500 ng/ml immediately prior to cranial neural crest migration was necessary to induce severe defects of the second visceral arch in a majority of embryos. Maximal increase in isotretinoin concentration to 16,000 ng/ml did not compensate for shorter exposure periods. These results suggest that to cause malformations of the visceral arches, the embryo must be exposed to isotretinoin for a minimum period of time regardless of the concentration of isotretinoin above the threshold.     

Inner ear malformations induced by isotretinoin in hamster fetuses.

Inner ear malformations induced in anotic hamster fetuses following maternal treatment with 50 mg/kg isotretinoin (13-cis-retinoic acid) on gestational day 8 are described. Computer-assisted three dimensional reconstruction was used. Two general types of defective vestibulocochlear development were seen. Defects were bilateral and correlated with extent of middle ear deficiency and severity of mandibular defects. In the more severely affected fetuses the inner ear was limited to an epithelial sac with occasional small projections, no apparent innervation and a correspondingly reduced otic capsule. In most of the fetuses examined the inner ear was less severely affected and was characterized by a reduction in the number of semicircular ducts and alterations in the size and shape of the cochlear duct. These defects are similar to those seen in a child with the isotretinoin embryopathy. Pathogenesis may result from a direct effect on otic epithelium or from faulty inductive interactions with the rhombencephalon or with periotic neural crest cells.     

Analytical method development and validation of mianserin hydrochloride and its metabolite in human plasma by LC-MS

Mianserin is a tetracyclic antidepressant drug and administered as racemate of R (-) and S (+) mianserin hydrochloride in a dose of 30-90 mg/day in divided doses. Liquid chromatography-mass spectroscopy (LC-MS) is a tool, which is widely used for determination of drug and their metabolites in biological fluids because of its high sensitivity and precision. Here we describe a liquid chromatography mass spectroscopy method for simultaneous determination of mianserin and its metabolite, N-desmethylmianserin, from human plasma using a liquid-liquid extraction with hexane:isoamylalcohol (98:2) and back extraction with 0.005 M formic acid solution. This method is specific and linear over the concentration range of 1.00-60.00 ng/ml for mianserin and 0.50-14.00 ng/ml for N-desmethylmianserin in human plasma. The lowest limits of quantification (LLQ) is 1.00 ng/ml for mianserin and 0.50 ng/ml for N-desmethylmianserin. Intraday and interday precision (%C.V.) is <10% for both mianserin and N-desmethylmianserin. The accuracy ranges from 94.44 to 112.33% for mianserin and 91.85-100.13% for N-desmethylmianserin. The stability studies showed that mianserin and N-desmethylmianserin in human plasma are stable during short-term period for sample preparation and analysis. The method was used to assay mianserin and its metabolite, N-desmethylmianserin, in human plasma samples obtained from subjects who had been given an oral tablet of 30 mg of mianserin.     

Teratogenic effects of alcohol and isotretinoin on craniofacial development: an analysis of animal models.

The teratogens alcohol and isotretinoin cause different patterns of facial dysmorphogenesis in the human. For isotretinoin the pattern is consistent with interference with the normal development of the cranial neural crest, particularly that destined for the second visceral arch. In vitro studies in the rat indicate that, at threshold levels of exposure to isotretinoin, the development of the second arch crest represents the most sensitive process of organogenic development. For alcohol, the facial abnormalities result from exposure very early in development, during the gastrulation process. There is no evidence that this is a peculiarly sensitive stage of development with respect to alcohol; animal studies indicate that other processes in the organogenic period are equally or more vulnerable. The emphasis given to the abnormal facial features in the fetal alcohol syndrome is considered a phenomenon associated with the exclusivity of syndromes.     

Isotretinoin teratogenicity in mouse whole embryo culture

Recent clinical observations strongly suggest that isotretinoin [13-cis-retinoic acid (cis RA)] is a human teratogen causing primarily heart and craniofacial malformations including ear and palatal defects. The purpose of the present study was to determine if cis RA could induce similar craniofacial malformations in mouse embryo culture. Day 8 CD-1 mouse embryos were cultured for 48 hours in rat serum in the presence or absence of various concentrations of cis RA dissolved in DMSO. DMSO by itself had no effect on embryonic development; however, cis RA at 2 X 10(-5) M (6 micrograms/ml) was clearly toxic. At 2 X 10(-6) M cis RA, growth retardation was minimal, and approximately one-third of the embryos exhibited very specific defects including a dramatic reduction in the size of the first and second visceral arches, which eventually give rise to the maxilla, mandible, and ear. Similar observations were also made with 4-oxo-13-cis RA, which is a major metabolite of cis RA in the mouse and human. These malformations would be expected to result in defects similar to those observed in the human, and preliminary observations suggest these defects are due to cis RA-induced inhibition of cranial neural crest cell migration. Using day-10 mouse embryos cultured for 48 hours in Waymouth's medium containing 50% fetal calf serum, we observed that cis RA at 2 X 10(-5) M produced a high percentage of embryos with limb defects and median cleft lip. Our results demonstrate that labeled cis RA enters the tissues of the embryo both in vivo and in vitro. Cis RA inhibited proliferation of the frontonasal mesenchyme cells in primary culture with 31% inhibition occurring at 2 X 10(-5) M cis RA.     

Gas chromatographic determination of 2- and 3-dechloroethylifosfamide in plasma and urine

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 μl of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 μg/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.     

Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 μg/ml in plasma and 0.1 μg/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01–2.5 μg/ml, and for the metabolite 0.01–1 μg/ml. In urine, the range for both compounds was 0.1–10 μg/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.     

Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction-liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific automated SPE-LC-MS-MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile-water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118, m/z 329-->196 and m/z 343-->196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1-200 ng/ml for I and II and 2-200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.     

Effects of isotretinoin on the behavior of neural crest cells in vitro

Isotretinoin (13-cis-retinoic acid), an anti-acne medication, has been found to cause severe birth defects which affect the craniofacial elements, ear, heart, thymus, and central nervous system. Many of these structures receive contributions from the cranial neural crest. Here, we examine the possibility that these teratogenic effects are due to disturbances in neural crest development. Cranial and trunk neural crest explant cultures were exposed to different concentrations of isotretinoin and the cell morphology was monitored at daily intervals. Treated neural crest cells often became rounded or spindle shaped, separated from their neighbors, and frequently detached from the substrate or clumped together. In contrast, neural tube cells and cardiac fibroblasts were relatively unaffected by the drug. These results suggest that isotretinoin selectively affects neural crest cells by decreasing their cell-substratum adhesion.     

Effect of culture age on the susceptibility of differentiating neuroblastoma cells to retinoid cytotoxicity

The cytotoxic effects of retinoids on neuroblastoma cells at various times during electrophysiological differentiation were evaluated. We used N1E-115, a clone of the murine neuroblastoma C1300 derived from the neural crest, and three retinoids: vitamin A (retinol), all-trans retinoic acid (tretinoin), and 13-cis-retinoic acid (isotretinoin). Differentiating N1E-115 cells exposed to retinoids at an isotretinoin EC(50) of 16 muM exhibited the greatest vulnerability in terms of cell death during a period (8 to 10 days) that was previously found to be the most sensitive for induction of gross malformations in rodents. This finding suggested possible similarities between the in vivo and in vitro retinoid mechanism(s) of action. The greatest period of vulnerability to retinoid cytotoxicity was also found to coincide with the rapid resting membrane potential (V(m)) development period, suggesting a linkage between neuronal V(m) and/or electrical excitability development and vulnerability to retinoid cytotoxicity. (c) 1996 John Wiley & Sons, Inc.     

Determination of unbound ticagrelor and its active metabolite (AR-C124910XX) in human plasma by equilibrium dialysis and LC-MS/MS

Ticagrelor is the first direct acting reversibly binding oral platelet P2Y(12) receptor antagonist. The parent molecule and the main metabolite (AR-C124910XX) are both able to block adenosine diphosphate-induced receptor signaling with similar potency. Drug binding to plasma proteins reduces free drug available for pharmacologic activity. Therefore, assessing unbound drug is important for interpretation of pharmacokinetic/pharmacodynamic findings. This paper describes the development and validation of an equilibrium dialysis/LC-MS/MS method for measuring unbound ticagrelor and AR-C124910XX in human plasma. Plasma samples (200μl) were dialysed against phosphate buffered saline (37 °C, 24h) in 96-well dialysis plates to separate unbound analytes. Drug-protein binding alterations during dialysis were minimized by maintaining physiologic conditions (pH 7.4, 37 °C). Ticagrelor and AR-C124910XX were quantified in dialysates (unbound fraction), retentates and plasma (total concentration) using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods. Calibration curves were established for the retentate and plasma (total concentration) in the ranges 5-5000 ng/ml (ticagrelor) and 2.5-2500 ng/ml (AR-C124910XX), and for the dialysate in the range 0.25-100 ng/ml (both analytes). Both ticagrelor and AR-C124910XX were highly protein bound (>99.8%), i.e. unbound fraction <0.2%. Yet, the methodology was successfully applied to determine unbound concentrations of ticagrelor and AR-C124910XX in clinical samples.     

Uptake and effect of praziquantel and the major human oxidative metabolite, 4-hydroxypraziquantel, by Schistosoma japonicum

After exposure to praziquantel in vitro at a concentration of 1 microgram/ml for 0.5-2 hr, amounts of praziquantel in Schistosoma japonicum varied from 2.1 +/- 1.2 to 3.7 +/- 1.6 ng/male worm and 1.3 +/- 1.2 to 2.2 +/- 1.5 ng/female worm during the time studied. At 30 micrograms/ml, praziquantel amounts were 11-33-fold higher. However, within 2 hr after removal from a medium containing 30 micrograms/ml praziquantel, 95% of the drug was released from the parasites. When S. japonicum worm pairs were incubated in vitro with 1, 10, and 30 micrograms/ml of 4-hydroxypraziquantel, the major human oxidative metabolite of praziquantel, 0.2 +/- 0.2, 3.8 +/- 1.3, and 7.4 +/- 1.3 ng/worm pair, respectively, were found after a 2-hr incubation. 15-30-fold lower than corresponding worm pair amounts of praziquantel. In vivo, when 4- or 5-wk S. japonicum-infected mice were treated orally with praziquantel (300 mg/kg), peak concentrations of praziquantel in plasma determined by high pressure liquid chromatography were 14.7 +/- 1.5 micrograms/ml (4-wk infection) and 16.7 +/- 2.8 micrograms/ml (5-wk infection) 15 min after treatment. Corresponding in vivo worm praziquantel amounts were 1.8 +/- 0.4 ng/male worm and 2.4 +/- 1.1 ng/female worm, respectively, in the 4-wk infection and 4.6 +/- 1.6 ng/male worm and 5.6 +/- 1.2 ng/female worm in the 5-wk infection. Peak plasma concentrations of 4-hydroxypraziquantel were similar but corresponding in vivo worm amounts were 1-20-fold lower, depending on the time after drug administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 13-cis-retinoic acid on hindbrain and craniofacial morphogenesis in long-tailed macaques (Macaca fascicularis)

Hindbrain and craniofacial development during early organogenesis was studied in normal and retinoic acid-exposed Macaca fascicularis embryos. 13-cis-retinoic acid impaired hindbrain segmentation as evidenced by compression of rhombomeres 1 to 5. Immunolocalization with the Hoxb-1 gene product along with quantitative measurements demonstrated that rhombomere 4 was particularly vulnerable to size reduction. Accompanying malformations of cranial neural crest cell migration patterns involved reduction and/or delay in pre- and post-otic placode crest cell populations that contribute to the pharyngeal arches and provide the developmental framework for the craniofacial region. The first and second pharyngeal arches were partially fused and the second arch was markedly reduced in size. The otocyst was delayed in development and shifted rostrolaterally relative to the hindbrain. These combined changes in the hindbrain, neural crest, and pharyngeal arches contribute to the craniofacial malformations observed in the retinoic acid malformation syndrome manifested in the macaque fetus.     

Sensitivity, specificity, and positive predictive value of multiple malformations in isotretinoin embryopathy surveillance

Isotretinoin causes serious birth defects in about 25% of babies exposed in the first trimester of pregnancy. Despite warnings about the drug's teratogenicity, cases of isotretinoin embryopathy continue to occur; more than 80 such cases have been reported since 1982. The true magnitude of the problem is unknown, however, and case estimates range to more than 1,000. The need for isotretinoin embryopathy (IE) surveillance is therefore great. Sixty-one known cases were evaluated to determine the sensitivity (proportion of cases with a given defect pattern) of various defect combinations. Using data from the Metropolitan Atlanta Congenital Defects Program for the period before isotretinoin was available, we evaluated the specificity (proportion of malformed infants without exposure who do not have the pattern of defects) for the various defect combinations. Ear malformations (microtia, anotia, absence or stricture of auditory canal, missing pinnae) have an associated sensitivity of 70.5% and a specificity of 99.5%. Ear defects combined with central nervous system (CNS) defects (microcephalus, hydrocephalus, reduction deformities of the brain) and cardiovascular (CVS) defects (conotruncal defects, aortic arch abnormalities) have an associated sensitivity of 19.7% and a specificity of 100.0%. The case definition of ear defects combined with either CNS or CVS defects maximizes both specificity (99.9%) and sensitivity (45.9%). The investigators are now evaluating the feasibility of using this pattern of defects to monitor for IE within a national monitoring program.     

Morphogenesis of isotretinoin-induced microcephaly and micrognathia studied by scanning electron microscopy

Isotretinoin ingestion during the first trimester of human pregnancy can induce malformations of the skull, ears, face, central nervous system, eyes, palate, lungs, circulatory system, limbs, and digits. A single oral dose of isotretinoin on day 8 of gestation in hamsters induces a similar syndrome of congenital malformation. The present study concerned scanning electron microscopic (SEM) observation of embryonic and fetal hamster craniofacial structures at 4, 8, 12, 24, 48, and 72 hr after administration of an oral dose of 50 mg/kg isotretinoin or an equivalent volume of the vehicle. The variability in development among control embryos recovered 4 hr after treatment precluded objective assessment of pathologic change by SEM at very early time points. Craniofacial damage was obvious within 8-12 hr of isotretinoin treatment, and it included hypoplasia of the maxillary and mandibular processes of the first branchial arch, a rudimentary second arch, and apparent collapse of the forebrain. Equivalent fusion between the lateral nasal process and the maxillary process and between the medial nasal process and the maxillary process in treated and control embryos accounts for the very low incidence of cleft lip observed in fetuses. The terminal microstomia was not associated with excessive merging or overgrowth of the first arch components. Hypoplasia of the first arch can account for retinoid-induced macrostomia and microstomia.     

Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro

Experiments were carried out to investigate putative beneficial effects of adding epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in chemically defined media. Presumptive zygotes (18 h post-insemination) were randomly assigned to culture treatments. In experiment 1, treatments involved additions of recombinant human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were seen in numbers of 4-cell stage embryos between groups. A concentration of 5 ng/ml EGF but not 1 or 25 ng/ml during embryo culture improved percentages of 4-cell stage embryos reaching blastocysts compared to the control (P<0.05). Numbers of inner cell mass (ICM) cells and trophoblast cells of day 8 blastocysts were similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no differences in numbers of 4-cell stage embryos between groups. When compared to controls, IGF-I treatments at 10 and 50 ng/ml improved proportions of 4-cell stage embryos that reached blastocysts (P<0.05). In experiment 3, numbers of ICM cells of day 8 blastocysts were significantly higher after being cultured with 50 ng/ml of IGF-I compared to those of the controls (P<0.05). No additive effect of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) was seen when results were compared to those following supplementation of the media with either EGF or IGF-I alone. In conclusion, both EGF and IGF-I could independently enhance bovine preimplantational development in chemically defined media and IGF-I but not EGF may play a mitogenic role during early bovine development.     

Detection and quantification of aripiprazole and its metabolite, dehydroaripiprazole, by gas chromatography-mass spectrometry in blood samples of psychiatric patients

Aripiprazole is a novel antipsychotic drug for the treatment of schizophrenia and schizoaffective disorders. In this study, a new method using gas chromatography-mass spectrometry (GC-MS) was developed and validated for the detection of aripiprazole and its main metabolite, dehydroaripiprazole, in plasma. Blood samples from seven psychiatric patients treated with aripiprazole (10-20 mg/day) underwent a solid-phase extraction (SPE) and N-methyl-N-trimethylsilytrifluoroacetamide (MSTFA) derivatization. The characteristic ions of mass spectra for aripiprazole and dehydroaripiprazole were m/z 306, 292, 218 and 304, 290, 218, respectively. Extraction recoveries from this method were 75.4% (n=5) for aripiprazole and 102.3% (n=5) for dehydroaripiprazole. The calibration curves of aripiprazole and dehydroaripiprazole were linear from 16 to 500 ng/ml (r(2)=0.999) and 8 to 250 ng/ml (r(2)=0.999), respectively. The respective limits of quantification (LOQs) for aripiprazole and dehydroaripiprazole evaluated in 0.5 ml of serum were 14.4 ng/ml and 6.9 ng/ml. Intra-assay and interassay precision and accuracy were within acceptable ranges. In this study, we also found that the mean trough concentrations in plasma at steady-state were 128.9 microg/l for aripiprazole and 30.1 microg/l for dehydroaripiprazole.     

Identification of 14-hydroxy-retro-retinol and 4-hydroxy-retinol as endogenous retinoids in rats throughout neonatal development

14-Hydroxy-retro-retinol was previously described as an in vivo and in vitro metabolite of retinol. Furthermore, the retinoid 4-hydroxy-retinol was identified as an endogenous occurring retinoid in the amphibian organism and an in vitro metabolite of retinol. We describe in the present study that 14-hydroxy-retro-retinol and 4-hydroxy-retinol are present in normal neonatal rat serum as endogenous occurring retinoids in normal non-vitamin A supplemented mammals (rats). Both retinoids were detected in serum and liver of neonatal rats at days 3 and 11 after birth. The respective concentrations at day 11 after birth were 41.8 +/- 2.8 ng/ml (serum)/ 104 +/- 6 ng/g (liver) for 4-hydroxy-retinol and 23 +/- 4.6 ng/ml (serum)/ 285 +/- 5 ng/g (liver) for 14-hydroxy-retro-retinol. Both retinoids could not be detected in adult rat serum and liver. From our experiments important physiological functions of these retinoids during postnatal development could be postulated.     

High-performance liquid chromatography determination of praziquantel enantiomers in human serum using a reversed-phase cellulose-based chiral stationary phase and disc solid-phase extraction

A sensitive HPLC method for the quantification of praziquantel enantiomers in human serum is described. The method involves the use of a novel disc solid-phase extraction for sample clean-up prior to HPLC analysis and is also free of interference from trans-4-hydroxypraziquantel, the major metabolite of praziquantel. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OJ-R) under isocratic conditions using a mobile phase consisting of 0.1 M sodium perchlorate–acetonitrile (66:34, v/v) at a flow-rate of 0.5 ml/min. Recoveries for R-(−)- and S-(+)-praziquantel enantiomers were in the range of 84–89% at 50–500 ng/ml levels. Intra-day and inter-day precisions calculated as R.S.D. were in the ranges of 3–8% and 1–8% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percent error were in the 0.2–5% and 0.3–8% ranges for both enantiomers, respectively. Linear calibration curves were in the concentration range 10–600 ng/ml for each enantiomer in serum. The limit of quantification of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using a UV detector set at 210 nm was 5 ng/ml (S/N=2).     

High-performance liquid chromatographic assay for xanomeline, a specific M-1 agonist, and its metabolite in human plasma

A reversed-phase high-performance liquid chromatographic assay (HPLC) was utilized for monitoring xanomeline (LY246708/NNC 11–0232) and a metabolite, desmethylxanomeline, in human plasma. Xanomeline, desmethylxanomeline and internal standard were extracted from plasma with hexane at basic pH. The organic solvent extract was evaporated to dryness with nitrogen and the dried residue was reconstituted with 0.2 M HCl-methanol (50:50, v/v). A Zorbax CN 150 × 4.6 mm I.D., 5-μm column and mobile phase consisting of 0.5% (5 ml/l) triethylamine (TEA) adjusted to pH 3.0 with concentrated orthophosphoric acid-tetrahydrofuran (THF) (70:30, v/v) produced consistent resolution of analytes from endogenous co-extracted plasma components. Column effluent was monitored at 296 nm/0.008 a.u.f.s. and the assay limit of quantification was 1.5 ng/ml. A linear response of 1.5 to 20 ng/ml was sufficient to monitor plasma drug/metabolite concentrations during clinical trials. HPLC assay validation as well as routine assay quality control (QC) samples indicated assay precision/accuracy was better than ±15%.