Chem. Res. Toxicol. 1993, 6, 180-187
180
Hepatic Cytochrome P450 2B-Type Induction by Ethyl/ Phenyl-Substituted Congeners of Phenobarbital in the Rat Raymond W. Nims,*tt Jia-Lin Syi,l David A. Wink,? Victor C. Nelson,$ Paul E. Thomas,li Collins R. Jones,l Bhalchandra A. Diwan,l Larry K. Keefer,t Jerry M. Rice,+ and Ronald A. Lubett Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21 702,Advanced Scientific Computing Laboratory, Chemical Synthesis and Analysis Laboratory, and Biological Carcinogenesis and Development Program, PRZIDynCorp, NCZ-Frederick Cancer Research and Development Center, Frederick, Maryland 21 702,and Department of Chemical Biology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855-0789 Received November 12,1992
As part of an investigation of the structural requirements for the induction, by phenobarbitaltype inducers, of a coordinate pleiotropic response consisting of increases in hepatic cytochrome P450 2B (P450 2B) activity, increases in other phase I and I1 enzyme activities, and liver hypertrophy, we have examined a series of analogues of phenobarbital in which the ethyl/phenyl substitution a t the sp3 carbon of the parent molecule was kept constant while the heterocyclic portion of the molecule was modified. The induction of hepatic P450 2B protein and ethoxy-, pentoxy-, and (benzy1oxy)resorufin 0-dealkylation activities, and epoxide hydration activity and livedbody weight ratio increase were examined in male F344/NCr rats fed the various congeners for 14 days a t doses equimolar to 500 ppm phenobarbital. Increases in the measured parameters were maximal in rats fed phenobarbital or 5-ethyl-5-phenylhydantoin. The responses to primidone or 2-ethyl-2-phenylsuccinimidewere approximately 65 % of maximal, while glutethimide yielded a response 50 % of maximal. Induction of this response in rats fed the ring-opened and decarboxylated analogues, (ethylphenylacety1)urea and 2-ethyl-2-phenylmacaused minimal increases lonamide, were 5.0); therefore, the percentages of the various compounds existing in the absorbable nonionized form within the stomach and small intestine are >99% and 159%, respectively (Table 111). Within the plasma, certain of the congeners (EPH, EPO, EPS, and PB) exist in the ionized state to varying extents (4-99%), resulting in a
184 Chem. Res. Toxicol., Vol. 6, No. 2, 1993
Nims et al.
Table 111. Physicochemical Properties of the Ethyl/Phenyl-Substituted Congeners % nonionized in physiological media congener"
pKab
stomach (pH 1.0)
EPO EPM EPAU GLUT EPS PRIM EPH
5.45 neutral (58) 11.22 9.2 (59) 8.76 (61) 13.0 (62) 8.51 (63) 7.41 (65)
>99 >99 >99 >99 >99 >99 >99 >99
PB
intestine (pH 5.3)c plasma (pH 7.4) 59 >99 >99 >99 >99 >99 >99 >99
1 >99 >99 >99 96 >99 93 51
1% P o c t b
areaidepth (nm)
area/depth*
0.58d 0.13d 1.71d 1.90 (60) 2.77d 0.91 (60) 1.34 (63,64) 1.34 f 0.13 (66-69)
10.5 11.1 14.8 12.9 12.3 11.4 11.4 12.5
1.4 1.5 2.3 1.9 1.8 1.5 1.6 1.7
a See Experimental Section for the chemical and trivial names of the drugs. Values shown were experimentally-determined (references given in parentheses). Underlined values were calculated, not experimentally determined. Virtual pH a t the luminal epithelial membrane (40). C. Hansch, personal communication.
net tendency of these agents to accumulate to a higher concentration in the blood than would result from equilibrium in the absence of ionization. The lipophilicities of the congeners (indicated by the log Pact values in Table 111)are sufficiently high in each case to facilitate passive diffusion of the un-ionized molecules through lipid membranes. From molecular modeling data (Table 111),the maximal molecular radii of the series of congeners were calculated and were found to range from 4.9 to 5.5 A, with the drugs being roughly spherical in conformation. The combined effects of the three physicochemical properties (low degree of ionization at the primary sites of absorption, relatively high lipophilicity, and similarity in molecular radius and shape) for these congeners suggest that the membrane permeability and therefore the rates and extents of absorption of these congeners may be expected to be roughly equivalent. The arealdepth and arealdepth2 ratios of the congeners, parameters which have previously been found to determine the isozyme specificity for substrates, inhibitors, or inducers of cytochrome P450 (46, 4 9 , were quite similar, ranging from 10.5 to 14.8 nm and from 1.4 to 2.3, respectively. Within these quite limited ranges of values, no apparent relationship with degree of P450 2B induction was apparent.
Discussion In this study, the effects of equimolar concentrations of a congenic series of compounds related to the prototype P450 2B inducer, PB, were examined in rats administered the drugs in the diet for 2 weeks. This duration was chosen to allow steady-state blood levels to be attained for each congener (requiring 7 half-lives of constant administration). For example, blood levels of PB, which has an elimination half-life of 9-11 h in the rat (15, 1 7 , 4 8 , 4 9 ) , should reach steady state within a few days of feeding, and we have observed in separate studies that serum PB concentrations attained after 1 and 2 weeks of administration of this drug at 500 ppm are equivalent (63 f 12 pM, n = 3, and 52 f 15 MM,n = 9, respectively). Since the elimination half-lives of certain of the congeners tested in the present study have not been determined in the rat, the use of 14 days of feeding allowed for the possibility that these agents might have half-lives longer than that of PB. While this experiment, involving dietary administration of equimolar concentrations of a series of compounds, was not designed to provide sufficient data to determine differences in induction potency, it was reasonable to expect that certain congeners might be identified which represent extreme cases (Le., compounds having minimal or great P450 2B-inducing ability). For instance, previous
-
screening of a number of barbiturate analogues (7,15-19) has consistently indicated that, within this congenic series, PB appears to be the most efficacious P450 2B inducer in the rat. One of the expectations of the present work was that, by testing a novel series of structural analogues, a compound superior in inducing ability to the prototype inducer, PB, might be identified. This is an important issue, since a major impediment to the identification of a cellular receptor for P450 2B induction has been the lack of a high-affinity/high-potency agonist (in analogy to TCDD) ( 1 4 , 5 0 ) . The results of this work indicate that the structure of the heterocyclic ring of PB may be substantially altered while retaining varying fractions of the inducing ability of the parent compound. These possible alterations include variations in the atoms of the six-membered heterocycle (PRIM and GLUT), elimination of one of the carbonyl groups to yield a five-membered heterocycle (EPH and EPS), and ring-opening of the heterocycle followed by decarboxylation (EPAU and EPM). EPH was previously shown by us to be approximately equivalent on a molar basis to PB in ability to induce P450 2B-specificO-dealkylation activities (24). The most interesting result of the present study may be the identification of a single substitution within the heterocycle (the replacement of the amido NH group of EPH with an oxygen to yield EPO) which appears to result in profound reduction in P450 2B-inducing ability. However, it is possible that the observed decrease in inducing ability may be due in some part to the relatively low pK, (-5.5) of the oxazolidinedione, which results in a high degree of ionization at physiological pH (-7.4). It has been shown previously by Stevenson and coworkers that PRIM is an effective inducer of cytochrome P450 and of P450-mediated monooxygenase activity (hexobarbital oxidase) in the rat when administered as four daily ip injections of 50 mg/kg body weight (52).This profound enzyme-inducing ability, confirmed in the present study, may be due in part to the formation of PB as a metabolic transformation product [- 15% conversion in the rat, the other major transformation product being EPM at -80% conversion (5211. Stevenson et al. also observed that EPAU, when administered at the same dose as PRIM, displayed more moderate induction of these P450 end points (51). The moderate ability (10-fold increase over control activity, -24% of the induction displayed by PB) of EPAU to induce P450 2B activities observed in the present study confirms their result and agreeswell with results previously reported by us (23). It has been demonstrated in crystallographic studies (53)that it is energetically favorable
Hepatic Cytochrome P450 2B-Type Induction
for EPAU to adopt a pseudocyclic conformation, a result which we now have confirmed using in vacuo molecular modeling. A similar pseudocycle is energetically favorable for EPM. The fact that a significant degree of inducing activity is retained by these compounds despite ringopening and decarboxylation could be rationalized by the formation of such a pseudocyclic conformation at a putative receptor site. Interestingly, EPAU has also been shown to be, on an equimolar basis, -7-fold more effective than PB in inducing the soluble cytochrome P450-dependent fatty acid monooxygenase of Bacillus megaterium (54). In these experiments, the individual enantiomers were similarly effective at inducing this monooxygenase, suggesting that there is little enantioselectivity involved in the induction mechanism. EPM was found to lack the ability to induce this bacterial monooxygenase, even when administered at a 4-fold higher concentration (54). The structureactivity relationships for the induction of the bacterial monooxygenase by barbiturate congeners also appear to differ from those observed in the rat. Specifically, in the bacterial system, inducing ability appears to correlate directly with lipophilicity, and the highly lipophilic congeners, secobarbital and thiopental, are 33- and 16-fold as effective on a molar basis as PB (55). This is in contrast to the results obtained with the rat, in which secobarbital (15, 17,19) and thiopental (17,18)appear to be weaker inducers than PB. In the rat, induction efficacy within the barbiturates has been found to be inversely correlated with lipophilicity (15) or to be independent of lipophilicity (17). These in vivo results must not be regarded as definitive, however, since the inducing abilities of the congeners examined were related to administered dose, rather than to active-site concentration. It is possible that the apparent differences in structureactivity relationships between the rat and B. megaterium may be rationalized at least in part by pharmacokinetics, which would be expected to play a major role in determining the steady-state drug concentrations attained within the hepatocytes. For instance, the structureactivity relationships for induction of P450 2H (the barbiturate-inducible form of P450 in the chicken) by C-5disubstituted barbiturates in cultured chick embryo hepatocytes indicate a direct correlation between inducing potency and lipophilicity (56). In this respect, the in vitro structure-activity relationships are quite similar to those obtained with B . megaterium. Efforts to characterize the structure-activity relationships of P450 2B induction in cultured rat hepatocytes, now in progress, also suggest that a direct relationship exists between P45O-inducing ability within a series of C-5-disubstituted barbiturates and lipophilicity.2 In the present study, by measuring food consumption, as well as through consideration of physicochemical properties of the congeners which play a major role in rate and extent of absorption from the gut, we have attempted to rule out differences in systemic dosage rate as a cause of the observed differences in P450 2B induction. This is important, since, under steady-state conditions, the serum levels of the various agents attained, and therefore the concentrations of drug attained within the hepatocyte and available to cause induction of P450 2B, depend upon (1)the systemic dosage rate and (2) the systemic elimi2
Peter Sinclair and Jacqueline Sinclair, personal communication.
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 185
nation rate of the congener. Despite the fact that the dosage rates for the various congeners may be assumed to have been roughly similar, the elimination rates may have varied markedly with subsequent effect upon steady-state serum concentrations. While no attempt was made to measure the steady-state concentrations for each compound examined in this study, testing of the pharmacodynamics of P450 2B induction by certain of the extreme cases identified, including PB, EPH, and EPO, has now been completed. On the basis of these studies (57),it has become clear that while to some extent the differences observed between the inducing abilities of these congeners relate to pharmacokinetics, a 10-fold difference in EC50 values exists between EPO and the other two congeners. While EPO is capable of eliciting a maximal P450 2B induction which is 75 % of that elicited by PB and EPH, the steady-state EPO concentrations required to elicit a half-maximal effect are 10-fold greater than the concentrations of PB or EPH required. The differences observed appear therefore to reflect actual differences in potency and efficacy, suggestingthat strict structural requirements may indeed exist for inducers of this form of P450. In summary, a novel congenic series of PB-like compounds has been characterized for ability to induce P450 2B protein and associated catalytic activities in the rat. The present study provides confirmation of earlier reports concerning certain of these analgues (EPAU, EPH, and PRIM) and, more importantly, allows the comparison of equimolar dose levels of these and other congeners in a single study performed systematically for the sole purpose of identifying relative inducing ability. N
Acknowledgment. We are grateful to D. Logsdon, C. Driver, G. Magruder, L. Riffle, and J. Carter for excellent technical assistance. This project has been funded at least in part with Federal funds from the Department of Health and Human Services under Contract N01-(20-74102 with Program Resources, Inc. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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