Phenobarbital labelling

In the case of amphetamine, the coupling is carried out directly from its primary amine [48]. Labeling of diphenylhydantoin was carried out by coupling onto 3-(2-aminoethyl)-5,5-diphenylhydantoin 36 [49], the latter amino derivative having been prepared by the method of O Neal [50]. In the case of phenobarbital, labeling is effected by coupling between m-aminophenobarbital 38 and cobaltocenium carboxylic acid hexafluorophosphate 39 in the presence of DCC [51]. Preparation of m-aminophenobarbital 38 was by electrochemical reduction of the nitro derivative 14 [51], whose synthesis is outlined in Scheme 8.9. 

In contrast to the metabolism of BA and BaP, the 5,6-dihydrodiols formed in the metabolism of DMBA by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are found to have 5R,6R/5S,6S enantiomer ratios of 11 89, 6 94, and 5 95, respectively (7.49 and Table II). The enantiomeric contents of the dihydrodiols were determined by a CSP-HPLC method (7.43). The 5,6-epoxide formed in the metabolism of DMBA by liver microsomes from 3MC-treated rats was found to contain predominantly (>97%) the 5R,6S-enantiomer which is converted by microsomal epoxide hydrolase-catalyzed hydration predominantly (>95%) at the R-center (C-5 position, see Figure 3) to yield the 5S,6S-dihydrodiol (49). In the metabolism of 12-methyl-BA, the 5S,6S-dihydrodiol was also found to be the major enantiomer formed (50) and this stereoselective reaction is similar to the reactions catalyzed by rat liver microsomes prepared with different enzyme inducers (unpublished results). Labeling studies using molecular oxygen-18 indicate that 5R,68-epoxide is the precursor of the 5S,6S-dihydrodiol formed in the metabolism of 12-methyl-BA (51). 

It should be remembered that this conversion is only approximate. Several other approximations have been used on the labels of certain tablets. For example. Saccharin tablets from Eli Lilly Company shows V2 gr (32 mg) on its label whereas the phenobarbital tablets from the same company have 30 mg (Z2 gr) on its label. Similarly sodium bicarbonate tablets from Eli Lilly have 5 grs (325 mg) on its label and potassium iodide from the same company has 300 mg (5 grs) on its container label. In the present book, it is advised to use one grain equivalent as 65 mg. 

It has been observed that while normal, rabbit serum failed to bind labelled phenobarbital, the serum from immunized rabbits bound 75 to 80% of the added pentobarbital and there exists a linear relationship between 14C-phenobarbital and the concentration of added antibody. Besides, when variable quantities of 14C-pentobarbital are added to a constant quantity of antibody, there exists a linear relationship between added and bound 14C-phenobarbital as depicted in Figure 32.4. 

Figure 32.4 Percentage Inhibition of Labelled Phenobarbital Binding by Added Unlabelled phenobarbital.

T. L. Keimig and L. B. McGown, Micellar modification of the spectral, intensity and lifetime characteristics of fluorescein-labeled phenobarbital, Talanta 33, 653-656 (1986). 

The tissue distributions of [l- C]- and [2,3- 4C]aciy lonitrile (40 mg/kg) were compared in Wistar rats after intraperitoneal and oral administration (Sapota, 1982). The relative distributions of the two labelled forms were very similar and the principal locations of C were erythrocytes, liver and kidney. After oral administration, the rate of elimination from tissues was slower for [cyano- C -than for [l,2-vz v/- 4C]acrylonitrile. After gavage administration of 46 mg/kg bw [2-i4C]acrylonitrile to Fischer 344 rats, radioactivity was well absorbed from the gastrointestinal tract and distributed to all major tissues 24 h after dosing. The highest levels were found in the forestomach, blood and urinary bladder. Prior treatment of rats with phenobarbital had little effect on the pattern of distribution and excretion of 4C, but the CYP inhibitor SKF-525A caused marked changes, with less excretion (less than 40% in urine in 24 h compared with over 60% in... 

If a drug is a substrate of CYP3A, coadministration with St. John s wort, a CYP3A inducer, can decrease the systemic exposure and effectiveness. St. John s wort may be listed in the labeling along with other known inducers, such as rifampin, rifabutin, rifapentin, dexamethasone, phenytoin, carbamazepine, or phenobarbital, as possibly decreasing plasma levels. 

Phenobarbital, a well-known hypnotic and antiepileptic agent, is a good example of a labelled synthesis [25]. The number of steps necessary for the labelling depends on the position of labelling that is, labelling in the 2 position of the... 

Heme may also be broken down into products beyond and other than bilirubin. Animals treated with phenobarbital formed a 1 1 molecular ratio of bilirubin to CO as expected of controls, but with an increased amount of bilirubin over the control. However, with animals treated with AIA, a major fraction of the breakdown products of labeled heme found in the bile was not bilirubin [Landow et al., 93a]. With a large subcutaneous dose of AIA (400 mg/kg) a decrease of cytochrome P450 in the rat is observable already after 1 hour, as well as a rise in ALA-synthetase. 

A second fraction of ELB is that probably derived primarily from the breakdown of the cytochrome P450 of the liver as first proposed by Schmid et al. [95]. It has a half-life of less than 12 hours. In studies of the decay of labeled heme of microsomal cytochrome P4S0, Levin and Kuntzman [96] observed two kinds of cytochrome P450. One had a half-life of 8 hours, the other of 48 hours the latter perhaps represents cytochrome P448. With phenobarbital treatment the ratio of these two kinds in the rat was 3.8 1. With 3-methyl cholanthrene treatment the ratio was 1 1. 

Labeling of phenobarbital, a barbiturate used in the treatment of convulsions, was carried out by acylation of p-aminophenobarbital 15 by the acid chloride of cymantrene 11 (Scheme 8.8). Synthesis of 15 was performed by nitration of the benzene ring of phenobarbital [35] to give a mixture of m- andp-nitrophenobarbital 13 and 14 followed by reduction of p-nitrophenobarbital by hydrogenation in the presence of palladium on charcoal [36]. 

Treatment of animals with phenobarbital for 3—6 days increases the liver weight, the liver weight/body weight ratio, microsomal protein content, and the synthesis of microsomal protein. Induction with 3-methylcholanthrene is accompanied by a smaller increase in liver mass and increased synthesis of protein from labelled precursors. In addition to stimulating liva growth in normal animals, enzyme inducers increase the rate of liver regeneration following partial hepatectomy. 

A stimulus which alters the steady-state level of an endogenous cellular component may do so by influencing its rate of synthesis, its rate of break-down, or both. When administered to intact animals, phenobarbital or 3-methylcholanthrene increase (20-50%) the steady-state level of microsomal protein. Similarly, micro-somes from animals pretreated with phenobarbital or 3-methylcholanthrene incorporate radioactive amino acids into protein more rapidly than microsomes from control animals and this effect is blocked by co-administration of actinomycin-D. It was therefore assumed that the increased levels of microsomal protein and enzyme activity after inducers were the result of enhanced synthesis. However, turnover studies have revealed that phenobarbital in particular has a profound effect upon microsomal protein catabolism. Proteins of the endoplasmic reticulum were labelled by injection of radioactive amino acids and the rate at which radioactivity disappeared from the microsomes was compared in control and phenobarbital-treated animals. Assuming a comparable degree of isotope re-utilization in the two groups, this approach provides a relative measure of microsomal-protein turnover. In control animals, radioactivity of total microsomal protein decreases with time with a half-time of about 3 days. In phenobarbital-treated animals, however, there is a marked stabilization of microsomal protein so that almost no radioactivity is lost over a S-day period. The reduced protein catabolism is observed both in total microsomes and in a purified microsomal protein, NADPH cytochrome c reductase. Thus, repeated administration of phenobarbital to animals evokes an increase in... 

Further labeled pyrimidines such as [2- C]barbituric acid (22, R, R = H) and its derivatives such as [2- C]barbital (22 R R = Et), and [2- C]pentobarbital (22 R = Et, R = 1-methylbutyl) are readily available from cyclocondensation of [ CJurea and the respective diethyl malonates in the presence of sodium ethoxide or butoxide. For phenobarbital (R = Et, R = Phe), decarboxylation of the malonate component was observed as a main side reaction, so that the respective malonyl dichloride had to be employed in order to achieve satisfactory yields. Analogous syntheses using unlabeled urea and labeled malonate derivatives are described in Chapter 6, Section 6.5. 

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