Donald S. Davies

Clinical pharmacology and pharmacokinetics of clonidine

A 300‐µg oral dose of clonidine was administered to 5 normal volunteers and measurements of plasma concentration and effects upon blood pressure, heart rate, circulatory reflexes, sedation, and dry mouth were made for the following 8 hr. The plasma concentration rose to a peak of 1.02 ± 0.52 ng/ml (SD) at 90 min andfell with a mean half‐life of 12.7 hr. Blood pressure of the group fell from 111.0/77.0 to 87.2/60.4 after 3 hr and was 95.2/62.2 mm Hg at 8 hr. Heart rate in recumbency was slowed. Marked sedation and a fall in sativary flow followed the same time‐course as the plasma concentration. The cold pressor response was reduced but the Valsalva overshoot was tiftle affected.

Pharmacokinetics and concentration‐effect relationships of intravenous and oral clonidine

The kinetics of the disposition of intravenous and oral clonidine in five normotensive subjects have been determined. It is proposed that a two‐compartment model adequately describes the disposition of the drug. The drug is rapidly distributed (tllza = 10.8 ± 4.7 min) but slowly eliminated (t½β = 8.5 ± 0.9 hr). The bioavailability of oral clonidine in the tablets tested averaged 75.2% and 40 to 50% of the bioavailable dose is excreted unchanged in urine. Renal clearance of the drug showed considerable intersubject variation (1.82 ± 0.34 ml/min/kg) and exceed the calculated glomerular filtration rate in some subjects. Oral and intravenous clonidine induced significant falls in blood pressure (>20/15 mm Hg) in our normotensive subjects and consistently caused marked sedation and dryness of the mouth. Sedation and salivary flow correlated with plasma clonidine concentration over the range 0 to 4 ng/ml. Falls in blood pressure were related to plasma concentration to 1.5 to 2 nglml but at higher concentrations the hypotensive effect was attenuated.

Resistance to β-adrenoceptor stimulants (a possible explanation for the rise in asthma deaths)

1 Resistance to isoprenaline has been produced in man and dog by prolonged exposure to the same, or the pharmacologically similar compounds, terbutaline and isoetharine. 2 In guinea‐pigs, pretreatment with these agents increases asthma mortality provoked by histamine which suggests that this resistance may reduce the effectiveness of endogenous sympathetic drive to the bronchi. 3 Possible mechanisms by which the asthma death rate could be increased are discussed. 4 It is suggested that a drug‐induced cross resistance to endogenous sympathetic stimulation could have led to a deterioration of the asthmatic state in patients using the pressurized aerosols of sympathomimetic bronchodilators and that this might account for the rise in asthma death rate.

Mechanisms of cell death.

Two distinct morphological patterns of cell death have been recognized, termed necrosis and apoptosis. Apoptosis, or programmed cell death, occurs in both physiological and pathological conditions. It arises due to an elevation of cytosolic free calcium concentration resulting in activation of a nuclear endonuclease. Activated endonuclease produces oligonucleosome-length DNA fragments. This DNA cleavage can directly precipitate cell death. Both glucocorticoids and TCDD may induce apoptosis by production of a heat labile factor that mediates calcium influx whereas tributyltin causes the opening of calcium channels. Evidence that perturbation in calcium homeostasis is an important event in cell necrosis is becoming increasingly persuasive, but the events that propagate the lesion are still unclear. Despite evidence for cytoskeletal disruption, activation of degradative enzymes such as proteases and phospholipase A2 and stimulation of other enzymes such as poly (ADP-ribose) polymerase, the exact role that these play in cell killing is not resolved. Indeed, recently the radical dichotomy between apoptosis and necrotic cell death has come into question. It is clear that further work is required to determine the role played by some elements of the apoptotic process in chemically induced cell death.

The specificity of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in the rat is the inverse of that in man

The kinetics of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in rat and human liver microsomes have been compared. Quinidine is a potent inhibitor of debrisoquine 4-hydroxylase activity of human liver (IC50: 3.6 microM). However, its stereoisomer, quinine, is some 60 times less potent (IC50:223 microM). Both compounds are able to inhibit greater than 95% of 4-hydroxylase activity. In rat liver microsomes quinine is approximately 50 times more potent an inhibitor (IC50:2.4 microM) than quinidine (IC50:137 microM). Again, 4-hydroxylase activity is inhibited by greater than 95%. Inhibition of debrisoquine 4-hydroxylase activity by both quinine and quinidine in human and rat liver is competitive. Values of Ki for quinidine in human and rat were 0.6 microM and 50 microM, whereas with quinidine the Ki values were 13 microM and 1.7 microM, respectively. The data in this paper are consistent with 4-hydroxylation of debrisoquine in both rat and human liver catalysed by a specific form of cytochrome P-450. Although both quinidine and quinine are competitive inhibitors of debrisoquine 4-hydroxylase activity in rat and man, their potency is reversed. This suggests that the nature of the active site of cytochrome P-450dbl differs between the two species, and indicates that data on the specificity of this isoenzyme in the rat should be extrapolated to man with extreme caution.

CLONIDINE WITHDRAWAL IN HYPERTENSION: Changes in Blood-pressure and Plasma and Urinary Noradrenaline

Treatment was interrupted abruptly in 6 hypertensive patients receiving clonidine 0-45-5-4 mg daily. Blood-pressure rose to pretreatment levels within 24-48 h of withdrawal and was accompanied by insomnia, headache, flushing, sweating, and apprehension. These symptoms began 18-20 h after the last dose of clonidine. Plasma-noradrenaline levels and urinary catecholamine excretion increased 24-72 h after withdrawal of clonidine. The subjective symptoms were most prominent in patients on higher doses (greater than 1 mg/day) and in those who had previously been receiving treatment with other antihypertensive drugs. One patient on a very low daily dose (0-15 mg) of clonidine had no symptoms and no significant changes in blood-pressure or catecholamine production after drug withdrawal.

Species variation in the response of the cytochrome P-450-dependent monooxygenase system to inducers and inhibitors

1. In the safety evaluation of drugs and other chemicals it is important to evaluate their possible inducing and inhibitory effects on the enzymes of drug metabolism. 2. While many similarities exist between species in their response to inducers and inhibitors, there are also important differences. Possible mechanisms of such variation are considered, with particular reference to the cytochrome P-450 system. 3. Differences in inhibition may be due to differences in inhibitory site of the enzyme involved, which is not always the active site of the enzyme, in competing pathways or in the pharmacokinetics of the inhibitor. 4. Differences in induction could be due to differences in the nature of the induction mechanism, in the isoenzyme induced, in tissue- or age-dependent regulation, in competing pathways for the substrate or its products, or in the pharmacokinetics of the inducing agent. 5. Examples of each of these possible differences are considered, often from our own work on the P450 IA subfamily, and results in animals are compared with those in humans, where possible. 6. At present, the differences between species in their response to inducers and inhibitors make extrapolation to humans from the results of animal studies difficult, so that ultimately such effects should be studied in the species of interest, humans.

Distribution and induction of CYP3A1 and CYP3A2 in rat liver and extrahepatic tissues

Previously, we have shown that highly specific antibodies against cytochrome P450 enzymes can be produced by targeting a 5-amino acid sequence at the C-terminus. Although rat CYP3A1 and CYP3A2 share 89% amino acid sequence similarity, they differ by 3 out of 5 of their C-terminal residues. In an effort to produce antibodies specific to each form, rabbits were immunised with the peptides IITGS and VINGA, corresponding to the C-termini of CYP3A1 and CYP3A2, respectively. Both antibodies bound strongly to hepatic microsomal fraction from rats treated with pregnenolone 16 alpha-carbonitrile (PCN) in enzyme-linked immunosorbent assay. Binding of the anti-IITGS antibody was strongly inhibited by incubation with IITGS, but VINGA was 60 times less effective. Conversely, binding of the anti-VINGA antibody was inhibited by VINGA 100 times more effectively than IITGS. Similar inhibition of antibody binding was also found using immunoblotting. Immunoadsorption using the anti-IITGS antibody yielded a single protein from solubilised hepatic microsomal fraction from PCN-treated rats, which was recognised only by the anti-IITGS antibody. Both antibodies bound to single proteins in the liver which were increased following treatment with PCN, but only the anti-IITGS antibody recognised protein in the lung, small intestine, and kidney of untreated and PCN-treated rats. Also, the binding of the two antibodies to hepatic and extrahepatic microsomal fractions from uninduced and induced rats showed differences in the expression of proteins recognised by the two antibodies, providing further evidence of antibody specificity. Thus, the binding of anti-IITGS and anti-VINGA antibodies is mutually exclusive and consistent with specific binding to their target antigens, CYP3A1 and CYP3A2, respectively. Immunocytochemistry was used to determine the distribution of CYP3A1 and CYP3A2. In the liver of untreated animals, both CYP3A1 and CYP3A2 were found to be expressed in the centrilobular region. However, some CYP3A1 immunoreactivity was also detected in many, but not all, hepatocytes throughout the lobule. However, following treatment of rats with PCN, both CYP3A1 and CYP3A2 were found to be strongly expressed in hepatocytes throughout the lobule, although CYP3A2 showed greater expression in the centrilobular region. PCN treatment was also found to result in induction of CYP3A1 in specific regions of the small intestine, lung, and kidney.

Immunocytochemical localization of cytochrome P-450 in hepatic and extra-hepatic tissues of the rat with a monoclonal antibody against cytochrome P-450 c

The cellular distribution of cytochrome P-450 has been studied in the liver and a number of extrahepatic tissues in the rat by immunocytochemistry, using an antibody raised against cytochrome P-450 form c. Immunoreactive cytochrome P-450, most probably form c, was found in the proximal tubules of the kidney, in the Clara cells of the lung, and in the olfactory epithelium and Bowmans glands of the olfactory tissue, in addition to its location in the liver. Immunoreactive cytochrome P-450 was not found in the small intestine, the testes or the adrenal gland, although these organs are known to contain isoenzymes of cytochrome P-450. The use of antibody titration enabled the effects of phenobarbitone, beta-naphthoflavone and clofibrate on the content and distribution of immunoreactive cytochrome P-450 to be studied in both the liver and in the other organs discussed. Phenobarbitone induces epitope-specific cytochrome P-450 in the centrilobular cells of the liver but has no effect in any of the other tissues studied. Clofibrate is without effect on the levels of immunoreactive cytochrome P-450 in any of the tissues studied. In contrast, beta-naphthoflavone induces immunoreactive cytochrome P-450 in the periportal region of the liver, and also in the Clara cells of the lung, in the enterocytes of the small intestine and in the proximal tubules of the kidney. Of all of the tissues studied, in which immunoreactive cytochrome P-450 could be detected, only the olfactory epithelium failed to undergo enzyme induction following treatment with beta-naphthoflavone.

Biphasic O-deethylation of phenacetin and 7-ethoxycoumarin by human and rat liver microsomal fractions.

Human and rat liver microsomal fractions exhibit non-linear Michaelis-Menten kinetics in the O-deethylation of both phenacetin and 7-ethoxycoumarin. Comparison of various models indicated that the data were best described by a biphasic plot, which could be interpreted in terms of two populations of cytochrome P-450. The K(m)s of the high affinity phase of 7-ethoxycoumarin O-deethylase activity were 1.8 +/- 0.4 microM and 2.3 +/- 0.4 microM for human and rat respectively while the K(m)s of the low affinity phase were 205 +/- 20 microM and 237 +/- 59 microM in the two species respectively. V(max) of the high affinity phase of human 7-ethoxycoumarin O-deethylase activity was 96.9 +/- 19.0 pmol mg(-1) min(-1) and the activity of the corresponding phase in the rat was 2.7 times greater. The activities of the low affinity phase were 10-15 times greater than the respective activity of the high affinity phase. Rat and human also had similar values for the K(m)s of the two phases of phenacetin O-deethylase activity, around 5 microM for the high affinity phase and 300 microM for the low affinity phase. Total activity was very similar in the two species, 1500-1750 pmol mg(-1) min(-1) and the difference between the two phases of activity was only 2.5-fold in man and 10-fold in rat. Studies on the effects of the in vitro modifiers of monooxygenase activity alpha-naphthoflavone and metyrapone further supported the hypothesis that the two phases of O-deethylase activity represent two different forms or populations of cytochrome P-450.