Plasma levels of the methyl ester of 15-methyl PGF2α in connection with intravenous and vaginal administration to the human

After intravenous injection of the methyl ester of 15-methyl-PGF_2α the drug initially disappeared faster than the corresponding free acid, but still after one hour, about 1% of the active drug is circulating in plasma. Vaginal administration of single suppositories containing 1 mg of 15-methyl-PGF_2α methyl ester and determination of plasma levels using gas chromatography - mass spectrometry demonstrated that the highest plasma levels were reached after 1.5 - 3 hours.Vaginal suppositories were administered according to different dose schedules for induction of abortion and plasma levels of 15-methyl-PGF_2α and it's ester were determined. There seemed to be a gross correlation between given doses and obtained plasma level. The data will serve as basis for further development of vaginal delivery devices.     

Radioimmunological identification and measurement of cytidine 3′, 5′-monophosphate in rat tissues

A radioimmunoassay for cyclic CMP³ is presented. Separation of cyclic CMP from other cyclic nucleotides and conversion to its 2′ O-acyl derivative was found necessary to achieve the specificity and sensitivity required. Low but easily measurable concentrations of cyclic CMP were found in rat liver spleen and kidney. Rat pancreas contained relatively higher amounts. These are the first precise determinations of cyclic CMP concentrations in mammalian tissues.     

Identification, expression and tissue distribution of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase activity in the rat

We report the postnatal developmental profiles of N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase) in different rat tissues. This enzyme, which catalyses the activation of NeuAc to CMP-Neu5Ac, was detected in brain, kidney, heart, spleen, liver, stomach, intestine, lung, thymus, prostate and urinary bladder but not in skeletal muscle. Comparative analysis of the different specific activity profiles obtained shows that the expression of CMP Neu5Ac synthetase is tissue-dependent and does not seem to be embryologically determined. Changes in the level of sialylation during development were also found to be intimately related to variations in the expression of this enzyme, at least in brain, heart, kidney, stomach, intestine and lung.     

Differential binding of rat pituitary-specific nuclear factors to the 5′-flanking region of pituitary and placental members of the human growth hormone gene family

Summary Placental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (–140/–107) and proximal site (–97/–66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (–140/–116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer. However, the data also indicate that rat Pit 1 binds to human and rat pituitary growth hormone in a similar manner (two sites of interaction) and that the pattern of binding is distinct from the placental members of the hGH gene family. These data indicate that human Pit 1, unlike the rat equivalent, might distinguish these genes functionally (tissue-specifically) as well as structurally.     

Synthesis of P-Thioadenylyl (2′-5′) Adenosine and P-Thioadenylyl (2′-5′)-P-Thioadenylyl-(2′-5′)-Adenosine

Abstract

Dimer and trimer adenylates with 2′-5′ phosphorothioate linkages were synthesized via the phosphoramidite method using p-nitrophenyl-ethyl group for phosphate protection and followed by sulfur oxidation. The various diastereoisomers were separated and characterized.     

Evidence for the conformation about the C(5′)-O(5′) bond of AMP complexed to AMP kinase: Substrate properties of a vinyl phosphonate analog of AMP

A vinyl phosphonate analog of adenosine 5′-phosphate (AMP) was synthesized in which the CH₂OP system of AMP is replaced by CHCHP. The V_max values of this analog relative to AMP were 0.7% with rabbit muscle AMP aminohydrolase, 13.4% with rabbit muscle AMP kinase, and 6.6% with pig muscle AMP kinase. The vinyl analog of ADP produced by the kinases was a substrate of rabbit muscle pyruvate kinase. These results, together with substrate specificity properties at the AMP sites of the enzymes indicate that the C(4′)-C(5′)-O(5′)-P system of AMP is of trans character during conversion of AMP to ADP by pig or rabbit AMP kinase.     

Metabolism of 16,16-dimethyl-Prostaglandin F2α in the cynomolgus monkey and the human female

The prostaglandin analog, 16,16-dimethyl-prostaglandin F_2α, tritium labeled in the 9β position, was administered by intramuscular injection into female cynomolgus monkeys and intravenous injection into human female subjects. The same products were found in the urine of both the monkey and the human female. The structures of three metabolites appearing in the urine from both species were determined. The main metabolite was the dinor derivative of 16,16-dimethyl-PGF_2α, which generally represented about 65% of the excreted amount of radioactivity in the urine from the cynomolgus monkey and about 45% in the urine from the human female. The other two metabolites were an ω-hydroxy metabolite of dinor-16,16-dimethyl-PGF_2α and the tetranor derivative of 16,16-dimethyl-PGF_2α. Studies on the disappearance of 16,16-dimethyl-PGF_2α from the circulation in human females showed a much longer half-life of this compound than of PGF_2α.     

Effect of chronic ethanol administration on the rat pineal N-acetyltransferase and thyroxine type II 5′-deiodinase activities

Chronic ethanol intake resulted in a significant decrease in the rate of rat ponderal growth and an impaired nyctohemeral profile of pineal N-acetyltransferase (NAT) activity. In ethanol-treated animals, the onset of the nocturnal NAT increase is delayed by 2 hours when compared to control animals. Moreover, pineal NAT nocturnal peak was reached at 4 h (2 hours later than controls), while pineal type II thyroxine 5-deiodinase (5-D) nyctohemeral profile was not modified by ethanol administration. The effect of ethanol administration (12 weeks) on 5-D activity in different tissues was also studied. Ethanol induced a 5-D activity increase in hypothesis and brain frontal cortex, when compared to control animals. No change in 5-D activity is observed in either pineal gland, Harderian gland, or brown adipose tissue. Since basal values of 5-D activity in hypophysis or brain frontal cortex are particularly dependent on serum thyroxine (T₄) concentration, the effect of chronic ethanol administration on thyroid hormone levels was studied. Serum T₄ levels in ethanol-treated animals were significantly decreased when compared to controls at any time point studied. However, no change in serum 3,3,5-triiodothyronine (T₃) levels were found.     

Isolation and sequencing of the 5′ end of the rat microtubule-associated protein (MAP1B)-encoding cDNA

We have isolated and sequenced the 5′ end of the cDNA encoding the rat microtubule-associated protein 1B (MAP1B). We found that this region is highly homologous to the corresponding regions of the human [Lien et al., 22 (1994) 273–280] and mouse [Noble et al., J. Cell Biol. 109 (1989) 3367–3376] MAPIB genes. The combination of the sequence that we are presenting with the previously published sequence [Zauner et al., Eur. J. Cell Biol. 57 (1992) 66–74], represents the complete rat MAP1B cDNA coding sequence.     

UPLC–QTOF/MS-based screening and identification of the constituents and their metabolites in rat plasma and urine after oral administration of Glechoma longituba extract

Glechoma longituba is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo integrated metabolism of its multiple bioactive components remains unknown. In this paper, ultra-performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight (QTOF) and the MetaboLynx™ software combined with mass defect filtering (MDF) together provide unique high throughput capabilities for drug metabolism study, with excellent MS mass accuracy and enhanced MS^E data acquisition. This rapid automated analysis method was successfully applied for screening and identification of the constituents absorbed and metabolized studies of G. longituba extract after oral administration to rats. The results showed that 21 parent components of G. longituba extract were absorbed into the blood circulation of the rats and a total of 80 metabolites of 9 parent compounds were tentatively detected in vivo by their MS spectra obtained at low or high collision energy scan with the comparison of the authentic standards and literature data. The developed method was simple and reliable, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of G. longituba and to better clarify its action mechanism. This work suggests that the integrative metabolism approach makes a useful template for drug metabolism research of TCM.     

Cellular and cytokine responses of the human central nervous system to intracranial administration of tumor necrosis factor α for the treatment of malignant gliomas

To elucidate the role of tumor necrosis factor (TNF) as a biological response modifier, we studied cellular and cytokine responses of the central nervous system to TNF administered intracranially in a phase I clinical trial for patients with malignant gliomas. Six patients received injections of TNF (1.25×10³–10×10³ U/injection) into the tumor cavities, and regional fluids (RF) and lumbar cerebrospinal fluids (CF) were serially sampled before and after the injections. Recruitment of neutrophils occurred, mostly peaking 8 h after TNF injection, and fewer numbers of CD4⁺ T cells and monocytes/macrophages migrated, subsequently peaking at 24 h. The CF leukocytosis persisted for 48 h and was associated with an increased level of neutrophil chemotactic activity in the CF. This neutrophil chemotactic activity was attributed to interleukin-8 (IL-8) by HPLC. The level of IL-6 activity in the CF and RF consistently increased; beginning 2 h after TNF injection and reaching the maximum between 8 h and 12 h. It returned to the basal level within 48 h. IL-1 was detected in the CF of three patients, its level peaking at 8 h. Prostaglandin E₂ also increased after injection of TNF, peaking between 4 h and 12 h and then gradually decreasing. Transforming growth factor was found in all cases tested and one patient showed a significant change after TNF injection. IL-2 activity, interferon (INF) activity, IFN, and granulocyte/macrophage-colony-stimulating factor were not detected in the CF or RF. In conclusion, TNF is biologically effective in inducing migration of immune cells and generating multiple cytokine responses in the human central nervous system.     

Design and Synthesis of bis-carbamate Analogs of Cyclic bis-(3′-5′)-Diguanylic Acid (c-di-GMP) and the Acyclic Dimer PGPG

The bacterial second messenger cyclic bis-(3′-5′)-diguanylic acid (c-di-GMP) regulates diverse Gram-negative bacterial virulence functions. The pathways that control, or are controlled by, c-di-GMP suggest that c-di-GMP signaling systems may encompass potential drug targets. It is presently undetermined, however, whether up- or down-modulation of c-di-GMP signaling would be the desired therapeutic state. We addressed potential drug target validation by synthesizing nonhydrolysable carbamate analogs of both the cyclic dinucleotide and the acyclic (seco) dinucleotide. A molecular docking simulation of the carbamate isostere suggests that this analog is capable of assuming the correct conformation and pose at a c-di-GMP binding site.     

Synthesis,Cytotoxicity and Metabolism of the 2′,2′-Difluoro-Analogs of Deoxyadenosine (dFdA)and Deoxyguanosine (dFdG)

Abstract

2′,2′-Difluoro analogs of deoxyadenosine (dFdA) and deoxyguanosine (dFdG) were synthesized. The in vitro toxicity and metabolism of dFdA and dFdG was studied in human leukemia cell lines.     

Metabolism of N-(3′,5′-Dichlorophenyl)succinimide in Rats and Dogs

When N-(3′,5′-dichlorophenyl)succinimide (DSI)-carbonyI-¹⁴C and –pheny-³H were each orally administered to rats, regardless of the label site, most of the dose was readily eliminated. There was no difference in the excretion rate between male and female rats. No radioactive residues were detected in tissues and organs 24 hr after dosing. Urinary metabolites consisted of N-(3′,5′-dichlorophenyl) succinamic acid (DSA), N-(3′,5′-dichlorophe-nyl) malonamic acid (DMA), N-(3′5’-dichlorophenyl)-2-hydroxysuccinamic acid (2-OH-DSA) and 2-OH-DSA derivatives. In dogs, most of the administered dose was excreted in equal amounts in urine and feces. 2-OH-DSA derivatives were main urinary metabolites and most of fecal radiocarbon was due to intact DSL. The results of this study indicate that DSI is a biodegradable compound which is unlikely to leave any persistent residues in animals.

The degradation of DSI to DSA was mediated by an arylamidase-type hydrolase, which was present in the microsomal fraction of rabbit liver. The enzyme activity was found in livers and kidneys of four animal species tested. Depending on the animal species, the enzyme appears to be important for the metabolism of DSI.     

In vivo processing of CXCL12α/SDF-1α after intravenous and subcutaneous administration to mice

CXCL12α has been shown to be selectively processed at the N‐ and C‐termini in blood and plasma in vitro. In order to study the processing in vivo, several versions of CXCL12α were expressed and purified. The protein was administered either iv or sc to mice, and at different time points postadministration plasma was collected and analyzed. To detect modifications of the CXCL12α molecule in crude plasma a SELDI TOF‐MS‐based method was developed. Anti‐CXCL12 antibodies were immobilized on the SELDI chip and CXCL12α binding to the antibodies was detected by SELDI‐TOF‐MS. The protein was found to be processed both at the C‐ and N‐termini. The same processed CXCL12α forms as detected in vitro were found; however, in addition further processing was detected at the N‐terminus, where altogether seven amino acids were removed. At the C‐terminus the lysine was removed as has been seen in vitro, and no further processing was detected. The full‐length CXCL12α disappeared within minutes after administration, whereas the processed forms of the protein were detectable for up to 6–8 h postadministration. The same processed forms appeared after iv and sc administration, only the kinetics was different. When the shortest processed form detected in plasma, 7ΔN1ΔC‐CXCL12α, was administered directly, no further processed forms were detected. Interestingly, a version of CXCL12α containing a N‐terminal methionine was protected against N‐terminal processing in plasma in vitro; however, in vivo no protection was seen, the protein was processed in the same way as full‐length CXCL12α.