Oral bioavailability of 17 β-estradiol and prodrugs tested in rats,pigs and dogs

In this work, 15 prodrugs of 17β-estradiol have been synthesized and characterized, and the kinetic profile of the compounds has been studied in rats, pigs and dogs after oral administration. All 15 substances were investigated in the rat model. The five substances, showing the highest bioavailability in this model, were selected for studies in pigs, and the two most promising compounds in pigs were subsequently tested in dogs.In the rat model, an improvement of the oral bioavailability with a factor three or more was seen from six of the prodrugs. In the pig model, a bioavailability, 2.5-3.7 times higher than that of estradiol was seen from three of five prodrugs.The dog model appeared extremely sensitive in proving the prodrug effect. Compared with the parent compound, an improved bioavailability of approximately 30 times was observed from the two prodrugs tested. After administration of these substances, the ratio of estrone and estradiol in serum was 0.4-0.6. It can be concluded that in all three animal species, the most promising prodrug properties appeared from the 3-glutarate-17-succinate and 3-benzyl-succinate esters of estradiol.     

Pharmacokinetics of amosulalol,an α, β-adrenoceptor blocker,in rats,dogs and monkeys

1. The pharmacokinetics of an α,β-adrenoceptor blocker, amosulalol hydrochloride, were studied after i.v. and oral administration to rats, dogs and monkeys.

2. After an i.v. dose (1 mg/kg), the plasma concentration-time curve fitted a two-compartment open model with terminal half-lives of 2-5 h in rats, 21 h in dogs and 1-8 h in monkeys. The order of plasma clearances for amosulalol was: rats > dogs > monkeys.

3. After oral administration, the maximum plasma concentration was obtained at 0-5-1 h in rats (10-100mg/kg) and dogs (3-30mg/kg), and at l-7-2-7h in monkeys (3-10 mg/kg). A linear relationship between the area under the plasma concentration-time curve and dose administered was obtained for all three species. The systemic availabilities of the drug in rats, dogs and monkeys were 22-31%, 51-59% and 57-66%, respectively.

4. After repeated oral administration (10 mg/kg) to dogs for 15 days, the pharmaco-kinetic parameters did not differ significantly from those on the first day.     

Genotoxicity assessment of β-caryophyllene oxide

β-caryophyllene oxide is a biciclic sesquiterpene, occurring naturally in essential oils from various medicinal and edible plants and used as a flavouring agent. Due to its potential hazardous chemical structure, the European Food Safety Authority reported to be pending a safety assessment for this compound. Here, this flavouring agent was tested for its mutagenic effect in the Ames test and micronucleus assay. Furthermore, considering that the penetration of a substance through phospholipid bilayers is determinant for its activity, the ability of β-caryophyllene oxide to be absorbed into cells was evaluated by differential scanning calorimetry (DSC) using multilamellar vesicles of dimyristoylphosphatidylcholine as a biomembrane model. β-caryophyllene oxide was found to be devoid of mutagenic effect, both at gene level (frameshift or base-substitution mutations), and on chromosome (clastogenicity and aneuploidogenicity). Results of DSC analysis highlighted that the substance was strongly absorbed through the membrane bilayer. Present results show that β-caryophyllene oxide, although absorbed through cell membranes and in spite of its potentially reactive chemical structure, is devoid of genotoxic effects, inducing neither point mutations nor chromosomal damages. These negative genotoxic findings will be critical to the safety assessment of β-caryophyllene oxide as used as a flavouring/fragrance ingredient.     

Effects of β-adrenoceptor antagonists on alcohol drinking by alcohol-dependent rats

Rationale  

Alcohol-dependent animals display enhanced stress responsivity, reward thresholds, and alcohol self-administration during alcohol withdrawal, and some of these aspects of alcohol dependence may be mediated by activation of brain norepinephrine (NE) systems.     

The impact of sesquiterpenes β-caryophyllene oxide and trans-nerolidol on xenobiotic-metabolizing enzymes in mice in vivo

1. Sesquiterpenes, constituents of plant essential oil, are popular bioactive compounds due to the positive effect on human health, but their potential toxicity and possible herb-drug interactions are often omitted. In our in vivo study, we followed up the effect of p.o. administration of two sesquiterpenes β-caryophyllene oxide (CAO) and trans-nerolidol (NER) on various xenobiotic-metabolizing enzymes in mice liver and small intestine.

2. To spot the early effect of studied compounds, enzymatic activity and mRNA levels were assessed 6 and 24?h after single dose.

3. CAO and NER markedly increased cytochromes P450 (CYP2B, 3A, 2C) activity and mRNA levels in both tissues. Liver also showed elevated activity of aldo-ketoreductase 1C and carbonyl reductase after treatment. Contrary, sesquiterpenes decreased NAD(P)H:quinone oxidoreductase 1 activity in small intestine. Among conjugation enzymes, only liver sulfotransferase activity was increased by sesquiterpenes.

4. Our results document that single dose of sesquiterpenes modulate activities and expression of several xenobiotic-metabolizing enzymes.     

Disposition and metabolism of amosulalol hydrochloride,a new combined α- and β-adrenoceptor blocking agent,in rats,dogs and monkeys

1. The disposition and metabolism of amosulalol hydrochloride, a combined α- and β-adrenoceptor blocking agent, were studied in rats, dogs and monkeys.

2. After oral administration of [¹⁴C]amosulalol hydrochloride, the plasma concentration of radioactivity reached a maximum at 05 to 1 h in all species and declined with half-lives of about 2 h in both rats and monkeys, and of about 4 h in dogs. The ratios of unchanged drug to total radioactivity in the rat and dog plasma were 8 and 43% at 05 h after administration, respectively. The radioactivity in the rat tissues was high in the liver, kidney, blood and pancreas after oral administration.

3. Following oral dosage, the urinary excretion of radioactivity was 26-34% of the dose in rats, 45% in dogs and 46% in monkeys in 48 h. The biliary excretion after oral dosage amounted to 66% and 41% in rats and dogs, respectively.

4. Six metabolites were isolated and identified from the urine of rats and dogs. They were derived from one or two of the following pathways: I, hydroxylation of the 2-methyl group of the methylbenzenesulphonamide ring; II, demethylation of the o-methoxy group of the methoxyphenoxy ring; III, hydroxylation at the 4 or 5 position of the methoxy-phenoxy ring; IV, oxidative cleavage of the C—N bond yielding o-methoxyphenoxy acetic acid. Moreover, some metabolites were metabolized to glucuronide or sulphate.     

Pharmacokinetic drug evaluation of budesonide in the treatment of Crohn’s disease

Introduction: Crohn’s disease (CD) is a chronic inflammatory disorder that commonly affects the terminal ileum and proximal colon. Although systemic corticosteroids such as prednisone and methylprednisolone are widely used for treatment of CD, these agents have a high incidence of adverse drug reactions due to off-target effects. Budesonide is a locally acting corticosteroid with enhanced formulation properties that offer a superior therapeutic index in comparison to conventional members of the class.

Areas covered: This review focuses on budesonide for the treatment of CD. The pharmacological and pharmacokinetics of the drug are summarized, along with clinical efficacy and safety data. We also indicate the role of budesonide in therapeutic algorithms.

Expert opinion: Budesonide has an important role as an induction therapy in patients with mild to moderately active CD of the ileum and proximal colon. The most distinctive advantage of budesonide over conventional corticosteroids is a substantially reduced risk of corticosteroid-related side effects.     

Single- and 14-day repeat-dose toxicity of cross-linked β-cyclodextrin in rats

The present study was carried out to investigate the oral single and repeat toxicity of cross-linked β-cyclodextrin (β-CD) made with adipic acid. Ten each of female and male rats were orally administered a 5000 mg/kg single dose. At this dose, mortality or macroscopic changes of internal organs were not observed. Dose levels 500, 1000, or 2000 mg/kg of β-CD were given daily to 5 each of female and male rats by oral gavage for 14 days. Body and organ weights were not significantly different during the study (P < .05). Hematology and clinical chemistry did not show any toxicity from the cross-linked β-CD. Macroscopic or microscopic changes were not observed between the controls and the treated rats. Based on these results, the cross-linked β-CD did not produce any signs of toxicity or other adverse effects at dose levels up to 2000 mg/kg per d for 14 days.     

Preclinical Pharmacokinetic Evaluation of β-Lapachone: Characteristics of Oral Bioavailability and First-Pass Metabolism in Rats

β-Lapachone has drawn increasing attention as an anti-inflammatory and anti-cancer drug. However, its oral bioavailability has not been yet assessed, which might be useful to develop efficient dosage forms possibly required for non-clinical and clinical studies and future market. The aim of the present study was thus to investigate pharmacokinetic properties of β-lapachone as well as its first-pass metabolism in the liver, and small and large intestines after oral administration to measure the absolute bioavailability in rats. A sensitive HPLC method was developed to evaluate levels of β-lapachone in plasma and organ homogenates. The drug degradation profiles were examined in plasma to assess the stability of the drug and in liver and intestinal homogenates to evaluate first-pass metabolism. Pharmacokinetic profiles were obtained after oral and intravenous administration of β-lapachone at doses of 40 mg/kg and 1.5 mg/kg, respectively. The measured oral bioavailability of β-lapachone was 15.5%. The considerable degradation of β-lapachone was seen in the organ homogenates but the drug was quite stable in plasma. In conclusion, we suggest that the fairly low oral bioavailability of β-lapachone may be resulted from the first-pass metabolic degradation of β-lapachone in the liver, small and large intestinal tracts and its low aqueous solubility.     

Stress induced desensitization of lymphocyte β-adrenoceptors in young and aged rats

The effects of different times of immobilization stress on intact lymphocyte β-adrenoceptors and plasma corticosterone were compared in 3-month and 24-month-old rats. In young animals after 30 min restraint ³H-dihydroalprenolol specific binding was significantly reduced (61% of control value) and plasma corticosterone significantly raised (186% of control). The effect on β-adrenoceptors was due to changes in receptor number (Bmax) without any effect on affinity (K_D). In aged rats both effects were only seen after 180 min restraint and were less pronounced. Isoproterenol treatment in vitro reduced β-adrenoceptors on lymphocytes. This effect was less pronounced in lymphocytes from aged rats. Corticosterone in vitro increased ³H-dihydroalprenolol specific binding. We therefore suggest that the decrease of β-adrenoceptors reflects an adaptive response to the stress-induced catecholamine release and that corticosterobe could play a role in reversing this effect. This adaptive response to stress seems to be impaired in aged animals.     

Concomitant use of tramadol and venlafaxine – evaluation of antidepressant-like activity and other behavioral effects in rats

BackgroundThe aim of this study was to evaluate antidepressant-like effect (Porsolt test), locomotor activity and motor coordination of joint administration of tramadol (TRM) and venlafaxine (VEN) in rats.MethodsThe tests were performed on male Wistar rats after single and chronic treatment (7 and 14 days) with TRM intraperitoneally (ip) and VEN orally (po) administered once a day. The controls were given 0.5% carboxymethylcellulose (CMC) solution (0.5 ml per rat, ip and po).ResultsIt was found that combination of TRM (5 mg/kg ip) with VEN (20 mg/kg po) caused an increased antidepressant effect compared to TRM and VEN administered alone, with no effect on locomotor activity or motor coordination in rats, which may be of clinical significance.It was also observed that reduced time of active swimming of animals in Porsolt test with an increased dose (10 and 20 mg/kg) and time of administration (7 and 14 days) of TRM were correlated with a decreased locomotor activity in rats. It may indicate the development of tolerance to TRM’s antidepressant effect in rats during chronic treatment with doses higher than 5 mg/kg.ConclusionIt can be expected that combination of low doses of TRM and VEN could potentially be feasible and relatively safe in cases with acute pain with co-existing depression, however, further investigations are needed.     

Changes of electrocardiogram and hemodynamics in response to dipyridamole: In vivo comparative analyses using anesthetized beagle dogs and microminipigs

Microminipigs are expected as a novel animal model for cardiovascular pharmacological experiments. Since inherent vulnerability of coronary circulation of microminipigs has not been characterized, we performed dipyridamole-stress test to both microminipigs and beagle dogs, and compared the results. Dipyridamole in doses of 0.056 and 0.56 mg/kg were intravenously infused over 10 min (n = 4 for each animal). Dipyridamole decreased the systolic/diastolic blood pressures and double product in dogs as well as in microminipigs; but it did not significantly alter the heart rate or the global balance between the myocardial oxygen demand and supply in either animal. While organic coronary arterial stenosis was not detected in either animal, dogs have well-developed epicardial intracoronary networks unlike microminipigs. Like in humans, dipyridamole did not affect the ST segment of microminipigs, whereas it substantially depressed that in dogs. The results indicate the onset of subendocardial ischemia by dipyridamole in dogs may be partly associated with their well-developed native coronary collateral channels. Microminipigs would be more useful to evaluate the drugs which may affect the coronary circulation in the pre-clinical study than dogs.     

Integrated TK–TD modeling for drug-induced concurrent tachycardia and QT changes in beagle dogs

Drug-induced cardiotoxicity, including tachycardia and QT prolongation, remains a major safety concern that needs to be identified and its risk mitigated in early stages of drug development. In the present study, an integrated toxicokinetic–toxicodynamic (TK–TD) modeling approach within a nonlinear mixed-effect modeling framework is applied to investigate concurrent abnormal heart rate and QT changes in three beagle dogs, using a Novartis internal compound (NVS001) as the case example. By accounting for saturable drug absorption, circadian rhythms, drug-effect tolerance, and nonlinear rate-dependency of QT interval, the dynamic TK–TD model captures the experimentally observed drug effects on heart rate and QT interval across a wide dosing range of NVS001 in beagle dogs. Further analyses reveal that the NVS001-induced QT prolongation observed in the low-dose groups is potentially caused by direct drug inhibition on the hERG channel, while the apparent QT shortening in the high-dose groups may be due to strong rate-dependency of QT at high heart rates. This study also suggests that the TK–TD model can be used to identify direct drug effects on the non-rate-dependent QT component by dissociating QT changes from tachycardia and deriving a new QT correction method. The integrated TK–TD model presented here may serve as a novel quantitative framework for evaluating drug-induced concurrent changes in heart rate and QT to potentially facilitate preclinical and clinical safety studies.     

Safety and pharmacokinetic studies of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol or γ-tocopherol in beagle dogs

Abstract

The safety and pharmacokinetic profile of liposomal formulations containing combinations of the antioxidants α-tocopherol, γ-tocopherol or N-acetylcysteine in beagle dogs was examined. Each group consisted of beagle dogs of both genders with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (330?mg/kg DPPC, EL), and test groups receiving liposomes prepared from DPPC lipids with (i) N-acetylcysteine (NAC) (60?mg/kg NAC [L-NAC]); (ii) NAC and α-tocopherol (αT) (60?mg/kg NAC and 25?mg/kg α-tocopherol [L-αT-NAC]) and (iii) NAC and γ-tocopherol (60?mg/kg NAC and 25?mg/kg γ-tocopherol (γT) [L-γT-NAC]). The dogs in the control group (EL) and three test groups exhibited no signs of toxicity during the dosing period or day 15 post treatment. Weight gain, feed consumption and clinical pathology findings (hematology, coagulation, clinical chemistry, urinalysis) were unremarkable in all dogs and in all groups. Results from the pharmacokinetic study revealed that the inclusion of tocopherols in the liposomal formulation significantly increased the area under the curve (AUC) and β-half life for NAC; the tocopherols had greater impact on the clearance of NAC, where reductions of central compartment clearance (CL) ranged from 56% to 60% and reductions of tissue clearance (CL₂) ranged from 73% to 77%. In conclusion, there was no treatment-related toxicity in dogs at the maximum feasible dose level by a single bolus intravenous administration while the addition of tocopherols to the liposomal formulation prolonged the circulation of NAC in plasma largely due to a decreased clearance of NAC.     

Disposition in rats and mice of chloropractolol,a new β-blocking agent

1. The disposition of a new β-blocking agent, chloropractolol, labelled with ¹⁴C and with ³H, has been studied in rats and mice.

2. After i.v. administration to the rat (25 mg/kg) of a mixture of [¹⁴C]- and [³H]chloropractolol, urinary and faecal excretion, biliary elimination and tissue distribution were similar for the two labelled species. However, 3% of the dose was recovered as ¹⁴CO₂.

3. Chloropractolol and/or metabolites did not show high cardioselectivity. Uptake by heart of the two labelled species was very close to that found for [¹⁴C]practolol and [¹⁴C]propranolol.

4. Preliminary metabolic studies using h.p.l.c. have shown that in the rat chloropractolol is extensively metabolized, which contrasts with the more limited metabolism of practolol in the same species. Deacetylation is a minor route of metabolism. A structure for the main urinary metabolite is suggested.     

Bioavailability and in vivo release behavior of controlled-release multiple-unit theophylline dosage forms in beagle dogs, cynomolgus monkeys, and göttingen minipigs

To determine the usefulness of monkey as an animal model, bioavailability and in vivo release behaviors of theophylline (TP) after oral administration of controlled-release beads in dogs, monkeys, and minipigs were evaluated. Controlled-release beads were prepared using a centrifugal-fluid type granulator, that is, CF-granulator, and Ethylcellulose (EC) was used as controlled-release coating agent. Aqueous solution and EC-coated beads, which contained TP were orally administered to animals after at least 1-week intervals. In dogs and minipigs, their relative bioavailabilities of EC-coated beads were 33.1% and 47.0%, respectively, and in vivo TP release from EC-coated beads in the gastrointestinal tract of dogs and minipigs were not reflected in vitro data. In monkeys, relative bioavailability of EC-coated beads was 80.0% and the highest among the three species, and release amount of TP from EC-coated beads at 24 h after oral administration was 82.8% and 92.4%, which was almost correlated to in vitro data. Therefore, the discrepancy of the relative bioavailability in three species is considered to be due to the difference of in vivo release behavior of TP. The monkey may be useful animal model for bioavailability studies of controlled-release dosage forms of TP from the viewpoint of in vitro-in vivo release correlation.     

Chronic administration of ethanol on pituitary and hypothalamic β-endorphin in rats and golden hamsters

The effect of chronic exposure to ethanol on hypothalamic and pituitary endorphin levels of rats and golden hamsters was studied. In rats, chronic ethanol consumption caused a decrease in the level of immuno-reactive (i.r.) β-endorphin (β-EP) in the pituitary but an increase of i.r. β-EP in the hypothalamus. The Met-enkephalin level in the hypothalamus of ethanol-treated rats remained the same as the control. The body weights, as well as food and liquid intake, of ethanol-treated rats were observed to be lower than the controls. In golden hamsters, i.r. β-EP level in the hypothalamus and pituitary remained unchanged with chronic ethanol consumption. The body weight and liquid intake of golden hamsters also remained the same as the controls. Since the changes of pituitary and hypothalamic β-EP after ethanol administration were found only in rats but not in golden hamsters, it is likely that the effects of ethanol observed in rats are not specific.     

Duration and selectivity in β-adrenoceptor blocking action of a β-adrenoceptor blocking drug,D-32 in conscious dogs

Summary In conscious dogs, the selectivity and duration of -blocking activity, and serum concentration of a -blocking agent, D-32 [dl-1-tert-butylamino-3-(2,3-dimethylphenoxy)-2-propanol hydrochloride] was compared to that of propranolol, pindolol, atenolol and IPS-339 [dl-1-tert-butylamino-3-(9-fluorenylideneaminoxy)-2-propanol hydrochloride]. Ratios of doses causing a 50% inhibition of tachycardia to that on hypotension induced by isoprenaline were as follows: D-32 (0.69), propranolol (0.67), atenolol (0.03) and IPS-339 (6.3). Thus, present experiments indicate that, unlike atenolol and IPS-339, D-32, propranolol and pindolol are non-selective -adrenoceptor blocking agents. Atenolol and IPS-339, however, selectively blocked cardiac ₁, receptors and vascular ₂-receptor respectively, as would be expected. In an optimal dose range these two drugs can be used satisfactorily as a pharmacological tool for inhibiting responses mediated via the respective -receptors. After oral administration, the pharmacological half-life (time required for 50% recovery of -blocking action) was 15.8±4.5 h for propranolol (3 mg/kg), 21.8±6.4 h for D-32 (0.5 mg/kg), 30.5±3.1 h for atenolol (6 mg/kg) and 30–35 h for pindolol (0.2 mg/kg). The pharmacological half-life after i.v. administration was 4.4±0.7 h for propranolol (300 g/kg) and 5.9±0.4 h for D-32 (150 g/kg), whereas the serum half-life (time required for 50% decrease in serum concentration) of propranolol was 1.4h and that of D-32 was 1.3 h. The values for pharmacological half-life and serum half-life were significantly different. Thus, for determination of administration frequency and dosage of -adrenoceptor blocking drugs, not only pharmacokinetic but also pharmacological data (duration of action) are essential.     

Plasma concentration and vascular effect of β-endorphin in spontaneously hypertensive and Wistar Kyoto rats

Summary In order to find out whether -endorphin (-E) is involved in the development of hypertension, we performed two series of experiments. Firstly, spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto controls (WKY) were submitted to ether stress. Plasma concentrations of -endorphin-like immunoreactivity (-EI), adrenocorticotropin (ACTH) and -melanotropin (-MSH) were measured by radioimmunoassay. The basal concentration of -EI was similar in WKY and SHR, whereas WKY had higher levels of ACTH and lower levels of -MSH than SHR. In both strains acute stress enhanced the plasma concentration of -EI to the same extent and with a similar time-course. The increase of plasma -El coincided with a rise in ACTH but not -MSH. Gel chromatography of -EI revealed that plasma extracts contain similar amounts of -lipotropin- (-LPH) and -E-sized immunoreactive components, and that acute stress elevated both forms of -El. Secondly, isolated tail arteries of SHR and WKY were perfused and field stimulated with two pulses at 1 Hz. -E depressed stimulation-evoked vasconstriction with the same potency in both strains. Thus, basal and stress-induced levels of -EI did not differ in SHR and WKY. Moreover, in the tail artery of both strains the sensitivity of presynaptic opioid receptors towards -E was almost identical. If the -E sensitivity of these receptors in other arteries of WKY and SHR is also similar, a major role of the circulating peptide in the development of hypertension is rather unlikely.This work was partly supported by the Deutsche Forschungsgemeinschaft (SFB 325) Send offprint requests to B. Bucher at the above address     

Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.