What makes a pluripotency reprogramming factor?

Resetting differentiated cells to a pluripotent state is now a widely applied technology and a key step towards personalized cell replacement therapies. Conventionally, combinations of transcription factor proteins are introduced into a differentiated cell to convert gene expression programs and to change cell fates. Yet, the molecular mechanism of nuclear reprogramming is only superficially understood. Specifically, it is unclear what sets pluripotency reprogramming factors (PRFs) molecularly apart from other transcription factor molecules that induce, for example, lineage commitment in embryonic development. Ultimately, PRFs must scan the genome of a differentiated cell, target enhancers of pluripotency factors and initiate gene expression. This requires biochemical properties to selectively recognize DNA sequences, either alone or by cooperating with other PRFs. In this review, we will discuss the molecular make-up of the prominent PRFs Sox2, Oct4, Klf4, Esrrb, Nr5a2 and Nanog and attempt to identify unique features distinguishing them from highly homologous yet functionally contrasting family members. Except for Klf4, the consensus DNA binding motifs are highly conserved for PRFs when compared to non-pluripotency inducing family members, suggesting that the individual DNA sequence preference may not be the distinguishing factor. By contrast, variant composite DNA motifs were found in pluripotency enhancers that lead to a differential assembly of various Sox and Oct family members due selective protein-protein interaction platform. As a consequence, the cooperation of PRFs on distinctly configured DNA motifs may underlie the reprogramming process. Indeed, it has been demonstrated that Sox17 can be rationally engineered into a PRF by modulating its cooperation with Oct4. An in deep understanding of this phenomenon would allow rational engineering and optimization of PRFs. This way, the reprogramming efficiency can be enhanced and fine-tuned to generate optimal synthetic reagents for regenerative medicine.

Health-related quality of life measures and psychiatric comorbidity in patients with migraine.

BACKGROUND AND PURPOSE: The identification of factors associated to health-related quality of life (HRQoL) measures in patients with migraine has major implications in terms of prognosis and treatment. This study aimed at investigating associations between HRQoL and comorbid mood and anxiety disorders. METHODS: Consecutive adult outpatients with a diagnosis of migraine with or without aura were assessed using the Mini International Neuropsychiatric Interview (M.I.N.I.) Plus version 5.0.0 and the Migraine-Specific Quality-of-Life Questionnaire (MSQ). RESULTS: Data of 112 patients (82 females), 69 without aura, mean age 41.2 +/- 13.3 years were analyzed. According to the M.I.N.I., 50% patients had a lifetime or current DSM-IV diagnosis of mood or anxiety disorder. There was no between-groups difference in MSQ total and subscale scores in relation to the presence/absence of psychiatric comorbidity, independently whether that was current or lifetime. In the group of subjects with psychiatric disorders, age at onset of migraine correlated with MSQ-total (rho = -0.407 P = 0.002), and subscale scores (Role Function-Restrictive, rho = -0.397, P = 0.002; Emotional Function, rho = -0.487, P < 0.001). CONCLUSIONS: Our findings suggest that current and/or lifetime psychiatric comorbidities are not associated with HRQoL measures in patients with migraine. However, patients with migraine and psychiatric comorbidities may represent a specific subgroup deserving particular attention for targeted interventions.

Alterations of glial cell function in temporal lobe epilepsy.

PURPOSE: Comparison of extracellular K+ regulation in sclerotic and nonsclerotic epileptic hippocampus. METHODS: Measurements of K+ signals with double-barreled K+-selective reference microelectrodes in area CAI of slices from human and rat hippocampus, induction of increases in extracellular potassium concentration by repetitive alvear stimulation or iontophoresis. and block of inward-rectifying and background K+ channels in astrocytes by barium. RESULTS: In the CA1 pyramidal layer from normal rat hippocampus, barium augmented extracellular K+ accumulation induced by iontophoresis or antidromic stimulation in a dose-dependent manner. Similarly, barium augmented stimulus-induced K+ signals from nonsclerotic hippocampi (human mesial temporal lobe epilepsy). In contrast, barium failed to do so in sclerotic hippocampi (human mesial temporal lobe epilepsy, rat pilocarpine model). CONCLUSIONS: Our findings suggest that in areas of reduced neuronal density (hippocampal sclerosis), glial cells adapt to permit rather large increases in extracellular potassium accumulation. Such increases might be involved in the transmission of activity through the sclerotic area.

Effects of barium on stimulus-induced rises of [K+]o in human epileptic non-sclerotic and sclerotic hippocampal area CA1.

In the hippocampus of patients with therapy-refractory temporal lobe epilepsy, glial cells of area CA1 might be less able to take up potassium ions via barium-sensitive inwardly rectifying and voltage-independent potassium channels. Using ion-selective microelectrodes we investigated the effects of barium on rises in [K+]o induced by repetitive alvear stimulation in slices from surgically removed hippocampi with and without Ammon's horn sclerosis (AHS and non-AHS). In non-AHS tissue, barium augmented rises in [K+]o by 147% and prolonged the half time of recovery by 90%. The barium effect was reversible, concentration dependent, and persisted in the presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid [GABA(A)] receptor antagonists. In AHS tissue, barium caused a decrease in the baseline level of [K+]o. In contrast to non-AHS slices, in AHS slices with intact synaptic transmission, barium had no effect on the stimulus-induced rises of [K+]o, and the half time of recovery from the rise was less prolonged (by 57%). Under conditions of blocked synaptic transmission, barium augmented stimulus-induced rises in [K+]o, but only by 40%. In both tissues, barium significantly reduced negative slow-field potentials following repetitive stimulation but did not alter the mean population spike amplitude. The findings suggest a significant contribution of glial barium-sensitive K+-channels to K+-buffering in non-AHS tissue and an impairment of glial barium-sensitive K+-uptake in AHS tissue.

Excretion and metabolism of remikiren, a potent orally active inhibitor of primate renin.

1. Following intravenous administration of 14C-remikiren to the male rat, 78% of the administered radioactivity was recovered in faeces, indicating high biliary elimination. Of the 25 +/- 0.1% of the dose recovered in urine, the majority (16.5% of dose) was intact drug. 2. After oral administration to the male rat the urinary recovery was markedly reduced (8.5 +/- 2.0% of dose), and virtually all of the material was excreted as an inactive hydrolysis product. Intact drug was non-detectable, suggesting extensive first-pass metabolism. 3. Perfusion of isolated rat liver confirmed high biliary elimination, coupled with extensive metabolism. Although intact remikiren was the major component in bile (20% of the 'dose'), the majority of the radioactivity was recovered as a series of mono- and di-hydroxylated metabolites. 4. When screened against human renin, only one of the metabolites in bile and urine (mono-hydroxylated in the t-butyl side chain, and synthesized as Ro 44-0444) showed comparable activity to remikiren. The remaining ten metabolites tested were at least one order of magnitude less active than the parent drug. 5. In comparative in vitro studies Ro 44-0444 was formed by rat, but not human or cynomolgus monkey, liver microsomes. The primate microsomes also produced more of the remaining mono- and di-hydroxy products, suggesting that metabolites make little contribution to the oral activity of remikiren which is observed in these species in vivo.

Animal pharmacokinetics and metabolism of the new antimalarial Ro 42-1611 (arteflene).

Ro 42-1611 (arteflene) is a new synthetic structural analogue of yingzhaosu, a Chinese traditional herbal drug, now under development for treatment of malaria. The in vivo activity of arteflene in a mouse animal model was 4-5 fold higher after parenteral than after oral administration. Pharmacokinetics of the drug were investigated in mice, rats, dogs, marmosets and cynomolgus monkeys. Plasma concentrations of arteflene were determined using a specific HPLC-UV method; the limit of quantification was 45 ng/ml using 0.5 ml plasma. The oral bioavailability was very low and variable (0.6% in mice, 4-5% in rats, 2.5 +/- 1% in dogs, < or = 0.5% in marmosets and < 0.5% in cynomolgus) as expected from the high metabolic clearance and the relative short apparent half-life (1.4-4.7 h). However, a metabolite (MA) was observed in plasma of all species indicating that drug was absorbed but underwent extensive first-pass metabolism. MA was also detected in samples of human plasma, collected during an oral tolerability study in healthy volunteers. After incubation of 14C-arteflene with liver microsomes of mice, rats, dogs and humans, the same major metabolite was detected and both samples were identical to Ro 47-6936 which was chemically synthesized as a reference compound. The in vitro activity of Ro 47-6936 was tested against Plasmodium falciparum and found to be about 1/4 that of the parent drug. Therefore, this metabolite makes a significant contribution to the biological activity in vivo, partially explaining the high activity of arteflene after oral administration in spite of its low bioavailability. Moreover, comparison of the metabolic patterns from human, rat and dog microsomes indicated that the dog is an appropriate species for toxicological evaluations.

Biotransformation of moclobemide in humans.

The structure of the urinary metabolites formed after moclobemide administration in human was elucidated, and the pattern compared with that in the plasma. The metabolic pathways of moclobemide were also compared with those of structurally related substances. After oral moclobemide administration, on average 95% of the dose was recovered in the urine within 4 days, with a mean of 92% being excreted during the first 12 h. The drug is extensively metabolized: less than 1% of the dose was excreted unchanged. A total of 19 metabolites, accounting together for about 64% of the dose, was isolated and all metabolites accounting for more than 1% of the dose were identified. Consistent with other morpholine-containing compounds, metabolic pathways of moclobemide include mainly oxidative attack on the morpholine moiety, leading to a multitude of oxidation products. Four primary metabolic reactions were identified: morpholine N-oxidation, aromatic hydroxylation, morpholine C-oxidation and deamination. The major metabolites in urine are 4 carboxylic acids (M7A and M7B, M8, M9) that account for 49% of the dose. Only 2 metabolites (M3, M10) were found to be hydroxylated on the aromatic nucleus. They were excreted completely as conjugates of glucuronic and/or sulfuric acid. Conjugation in general, however, seems to be of minor importance in the overall biotransformation of the drug. The metabolite pattern in plasma was found to be qualitatively but not quantitatively similar to that observed in urine. Almost all of the main urinary metabolites were found in plasma as well. The unchanged parent compound and 2 primary oxidation products of the morpholine ring (M1, M15), which were present in urine only in trace amounts, could easily be detected in plasma.

Fully automated high-performance liquid chromatography. A new chromatograph for pharmacokinetic drug monitoring by direct injection of body fluids.

A new fully automated high-performance liquid chromatography is described which detects drugs from directly injected plasma (urine, saliva) without sample pretreatment. The apparatus consists of a programmable automatic sampling unit, which is connected via two alternating working pre-columns to an analytical column ("alternating pre-column sample enrichment"). The new device is able to operate with directly injected body fluids like an auto-analyzer and is especially useful for pharmacokinetic and clinical studies, where drug concentrations have to be determined from plasma, urine or saliva.

[Metabolism of Ro 21-5998 (mefloquine) in the rat (author's transl)]. / Metabolismus von Ro 21-5998 (Mefloquin) bei der Ratte.

After administration of 15 mg/kg 14C-Ro 21-5998/001 i.p. to rats, the metabolite patterns in feces, urine, bile and blood were compared. Metabolites from feces were identified by GC/MS, in all other cases by TLC. The main component in the feces consists of mefloquine (Ro 21-5998). In addition the acid Ro 21-5104, a derivative of mefloquine with a hydroxy group in the piperidine moiety (M 12), the alcohol Ro 14-0518 and a metabolite (M 4a), which is supposed to be a lactam, were shown to be present. The acid Ro 21-5104 is the main metabolite in the urine. The bile contains the parent compound Ro 21-5998, the acid Ro 21-5104 and the alcohol Ro 14-0518, partially as conjugates. The structurally investigated components account for about 40% of the administered dose. The blood contains the parent compound Ro 21-5998 and the acid Ro 21-5104 as main components. In comparing the various metabolite patterns, it can be summarized that that in urine is slightly different from those in bile (after hydrolysis of the conjugates), feces and blood which show more similarities between each other.

[Alterations in pharmacokinetics during toxicity tests (author's transl)]. / Pharmakokinetische Untersuchungen als Hilfsmittel zur Interpretation des Verlaufs von Toxizitätsprüfungen.

Some possible reasons are discussed which may lead to changes in the pharmacokinetics of a drug during long-term toxicity tests. Since most of these alterations have toxicological consequences, their evaluation can be helpful in the interpretation of toxicity studies. Furthermore, and altered pharmacokinetic profile may be a diagnostic tool for the detection of organ damage (e.g., eliminatory organs). In this paper three examples of the "radioactive test dose method" are shown. This method allows a direct comparison of the pharmacokinetics between pretreated animals and controls by administration of a single radioactive test dose to both groups.

[Ambroxol, comparative studies of pharmacokinetics and biotransformation in rat, rabbit, dog and man (author's transl)]. / Speziesvergleich in Pharmakokinetik und Metabolismus von NA 872 Cl Ambroxol bei Ratte, Kaninchen, Hund und Mensch.

Pharmacokinetics and biotransformation of trans-4-(2-amino-3,5-dibromo-benzylamino)cyclohexanol hydrochloride (ambroxol, NA 872 Cl) was studied using the 14C-labelled compound. Absorption after oral administration was found to be fast and complete. Elimination half-life of radioactivity in the blood was estimated as 20--25 h in rat, dog and man and as 2 h only in rabbit. This apparent elimination half-life is governed by the disposition of acidic metabolites of NA 872. In man and rabbit radioactivity is excreted almost completely into the urine, whereas in rat and dog biliary excretion is also observed. Routes of biotransformation are similar in all 4 species. NA 872 is metabolized by phase I reactions to NA 873 and finally to dibromoanthranilic acid. Phase II reactions with the parent compound and metabolites are observed mainly in man and rabbit.

[Ambroxol, studies of biotransformation in man and determination in biological samples (author's transl)]. / Ambroxol, Untersuchungen zum Stoffwechsel beim Menschen und zum quantitativen Nachweis in biologischen Proben.

15 mg trans-4-[2-amino-3,5-dibromo-benzyl)-amino]-cyclohexanol-hydrochloride (ambroxol, NA 872) was administered i.v. and orally to healthy volunteers. The metabolic pattern in urine and plasma was similar for both routes of administration. Biotransformation reactions are straightforward, yielding two major products of phage I reactions identified as 6,8-dibromo-3-(trans-4-hydroxycyclohexyl)-1,2,3,4-tetrahydro-quinazoline and 3,5-dibromo-anthranilic acid. These metabolites as well as the parent compound are also converted to conjugates, predominantly glucuronides. Quantification of unlabelled ambroxol in biological fluids is achieved by radiochemical derivatisation with 14C-labelled formaldehyde in imitation of the biotransformation.

[Pharmacokinetics and metabolism of trazodone in man (author's transl)]. / Pharmakokinetik und Stoffwechsel von Trazodone beim Menschen

After intravenous, intramuscular and oral administration of 25 mg 14C-labelled 2-(3-[4-(m-chlorophenyl)-1-piperazinyl]-propyl)-s-triazolo[4,3-a]pyridin-3-(2H)-one-hydrochloride (trazodone), plasma levels, elimination and metabolite pattern in plasma and urine were investigated. The plasma levels after all routes of administration are almost identical. The absorption of the compound is fast and complete. The elimination of radioactivity occurs in a biphasic manner with a half-life of 1 h for the earlier and 13 h for the second phase, no matter what application route had been chosen. The comparison of the plasma levels of fasted and non-fasted subjects shows a shift of the plasma maximum from 1.5 to 2.5 h following administration and a decrease in the maximum level of 30%. The radioactivity is excreted predominantly by renal processes (70-75% within 72 h). The main product in plasma is unchanged trazodone, whereas in urine it is found only in minute amounts. The radioactivity in urine is represented by conjugates that had formed after hydroxylation on the chlorophenyl residue (20%), by a dihydrodiol-metabolite of trazodone (15%) and by a carboxylic acid originating from oxidative cleavage of the parent compound (35%).

Bis-(p-hydroxyphenyl)-pyridyl-2-methane: The common laxative principle of Bisacodyl and sodium picosulfate.

After both oral and rectal administration in humans (4,4'-diacetoxy-diphenyl)-(pyridyl-2)-methane (bisacodyl, Dulcolax) and 4,4'-(2-pyridyl-methylene)-diphenol-disulfuric acid semiester disodium (sodium picosulfate, Laxoberal) are hydrolyzed to bis-(p-hydroxyphenyl)-pyridyl-2-methane (BHPM). In both cases BHPM is responsible for the laxative action. Experiments in rats and guinea pigs have shown that the hydrolysis of picosulfate, in contrast to that of bisacodyl, is attributable to the microorganisms of the intestinal flora.

[Pharmacokinetics and metabolism of the antiinflammatory agents S-H 766 in man]. / Pharmakokinetik und Metabolismus des Antiphlogistikums S-H 766 beim Menschen

Blood and plasma levels as well as urinary and fecal excretion were measured in humans after oral administration of radioactively labelled 4-[j-(2'-fluorobiphenylyl)]-4-hydroxycrotonic acid (S-H 766 MO). The radioactivity in the plasma reaches maximum values of about 10 mug eq./ml 1 to 2 h after application with either form. After repeated administration good agreement is found between the plasma levels measured and those simulated according to the pharmacokinetic parameters obtained after single application. The S-H 766 metabolites were investigated in blood and urine. The substance was found to undergo considerable metabolism, only approximately 2% being excreted in the urine unchanged. The conjugates, which constitute over 60% of the radioactivity of the urine, consist mainly of glucuronides and sulfates. The structure of the aglycones shows that the metabolism occurs along two pathways, by beta-oxidation of the aliphatic side chain into aryl acetic acids and by hydroxylation of the aromatic nucleus to phenolic compounds. It must be assumed that these biotransformations take place both simultaneously and successively.