Pharmacokinetics and tolerability of cinitapride in healthy Chinese volunteers: a randomized,open-label,single- and multiple-dose study

1. Cinitapride (CIN) is a drug for functional dyspepsia. The purpose of the study was to investigate the pharmacokinetics and tolerability of CIN in healthy Chinese volunteers.

2. A randomized, open-label, single- and multiple-dose study was conducted in 12 healthy volunteers. Three different doses of CIN (1, 2, 4 tablets) were given to six groups in the single-dose study, and one tablet (1?mg) of CIN was administered three times a day in the multiple-dose study. Blood samples were collected at predetermined time intervals after CIN dosing and analyzed by LC-MS/MS.

3. Eleven volunteers completed the study. After single dose, the C_max and AUC of plasma increased approximately linearly with dosage; no statistically significant differences were found in pharmacokinetic parameters between three dose groups. After multiple doses, there was no significant change in T_max and t_1/2 compared with the results from the single dose. After repeated doses, AUC_0-t and AUC_0-∞ were increased, while CLz/F slightly decreased. And no differences between male and female.

4. The pharmacokinetic parameters of this study were consistent with study results of non-Chinese subjects. Good tolerability was demonstrated in both single- and multiple-dose studies with dosage range from 1 to 4?mg in healthy Chinese subjects.

     

Rats and rabbits as pharmacokinetic screening tools for long acting intramuscular depots: case study with paliperidone palmitate suspension

1. Development of prodrug of 9-hydroxyrisperidone (paliperidone) long-acting intramuscular injection has enabled delivery over four-week time period with improved compliance.

2. The key aim of this work was to establish a reliable preclinical model which may potentially serve as a screening tool for judging the pharmacokinetics of paliperidone formulation(s) prior to human clinical work.

3. Sparse sampling composite study was used in rats, (Wistar/Sprague–Dawley (SD; n?=?10)) and a serial blood sampling study design was used in rabbits (n?=?4). Animals received intramuscular injection of paliperidone palmitate in the thigh muscle at dose of 16 (rats) and 4.5?mg/kg (rabbits). Samples were drawn in rats (retro-orbital sinus) and rabbits (central ear artery) and were analysed for paliperidone using liquid chromatography–mass spectrometry/ mass spectrometry (LC-MS/MS) assay. The plasma data was subjected to pharmacokinetic analysis.

4. Following intramuscular injection of depot formulation in Wistar/SD rats and rabbits, absorption of paliperidone was slow and gradual with median value of time to reach maximum concentration (T_max) occurring on day 7. The exposures (i.e. area under the curve (AUC; 0–28) days) were 18,597, 21,865 and 18,120?ng.h/mL, in Wistar, SD and rabbits, respectively. The clearance was slow and supported long half-life (8–10 days).

5. Either one of the two models can serve as a research tool for establishing pharmacokinetics of paliperidone formulation(s).

     

Dose proportionality and pharmacokinetics of dronedarone following intravenous and oral administration to rat

1. The aim of this study was to investigate the pharmacokinetic properties of dronedarone by using noncompartmental analysis and modeling approaches after intravenous and oral administration of dronedarone to rats.

2. Twenty-eight male Sprague-Dawley rats were randomly divided into four groups, and dronedarone was administered intravenously (1?mg/kg) and orally (5, 10 and 40?mg/kg) based on a parallel design. Blood samples were collected before and 0.083 (intravenous administration only), 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12 and 24?h after drug administration. The plasma concentration of dronedarone was determined by using LC-MS/MS.

3. The oral bioavailability of dronedarone was evaluated as approximately 16% in rats, similar to that in humans. The assessment of dose proportionality by using the power model showed that AUC_inf increased in a dose-proportional manner, whereas AUC_24h and C_max exhibited a lack of dose proportionality over the dose range between 5 and 40?mg/kg. The two-compartment model, with first-order absorption and elimination rate constants, was sufficient to explain the pharmacokinetics of dronedarone with biexponential decay.

4. These findings will help to understand the pharmacology of dronedarone to develop the new formulation and therapeutics optimization linked to pharmacokinetic/pharmacodynamic study.

     

Expression and activities of sulfotransferase in rat brain

1. Sulfotransferase (SULT) has been found in the brain; however, the details of its function remain unclear. The present study aimed to elucidate the regional differences in the expression of SULT1 and SULT2 mRNA and SULT activities in the eight functional regions of the rat brain (cerebellum, cortex, hippocampus, medulla oblongata, midbrain, olfactory bulb, striatum, and thalamus).

2. All SULT1 isoforms were detected in the medulla oblongata and thalamus. SULT2A1 mRNA was not observed in any of the eight regions, whereas SULT2B1a and SULT2B1b were found in all regions. The SULT2B1b mRNA expression level in the medulla oblongata was 1.7-fold higher than that in the liver. The sulfonation of p-nitrophenol and pregnenolone was detected in all regions. The kinetics of p-nitrophenol sulfonation in the cerebellum fitted to the substrate inhibition model (K_m?=?37.6?nM, V_max?=?2.72?pmol/min/mg, V_inh =?1.60?pmol/min/mg, and K_i?=?0.87?μM). The pregnenolone sulfonation also exhibited substrate inhibition kinetics (K_m?=?0.99?μM, V_max?=?1.53?pmol/min/mg, and K_i?=?54.67?μM).

3. We clarified that SULT1 and SULT2 were expressed and had metabolizing capacities in the rat brain, suggesting that brain SULTs may be involved in metabolism of endogenous compounds and drugs.

     

Effect Of epigallocatechin-3-gallate on the pharmacokinetics of amlodipine in rats

1. This study investigates the effect of epigallocatechin-3-gallate (EGCG), a major ingredient of green tea, on the pharmacokinetics of amlodipine in rats.

2. The pharmacokinetics of orally administered amlodipine (1?mg/kg) with or without EGCG pretreatment (30?mg/kg/day for 10?days) were investigated. Plasma concentrations of amlodipine were determined by using a sensitive and reliable liquid chromatography with tandem mass spectroscopy (LC-MS/MS) method. The effects of EGCG on the metabolic stability of amlodipine were investigated by using rat liver microsome incubation systems.

3. The results indicated that when the rats were pretreated with EGCG, the C_max of amlodipine increased from 16.32?±?2.57 to 21.44?±?3.56?ng/mL (p?<?0.05), the T_max decreased from 5.98?±?1.25 to 4.01?±?1.02?h (p?<?0.05), and the AUC_0–t increased from 258.12?±?76.25 to 383.34?±?86.95?μg h L^?1 (p?<?0.05), which suggested that the pharmacokinetic behavior of amlodipine was affected after oral co-administration of EGCG. Additionally, the metabolic half-life was prolonged from 31.3?±?5.6 to 52.6?±?7.9?min (p?<?0.05) with the pretreatment of EGCG.

4. It can be speculated that the drug-drug interaction between EGCG and amlodipine might occur, which might have resulted from the metabolism inhibition of amlodipine by EGCG when they were co-administered.

     

Investigation of disposition for TAK-448, a synthetic peptide of kisspeptin analog,in rats and dogs using the radiolabeled TAK-448 suitable for pharmacokinetic study

1. Disposition of 2-(N-acetyl-d-tyrosyl-trans-4-hydroxy-l-prolyl-l-asparaginyl-l-threonyl-l-phenylalanyl) hydrazinocarbonyl-L-leucyl-N^ω-methyl-l-arginyl-l-tryptophanamide monoacetate (TAK-448, RVT-602), a synthetic kisspeptin analog, was investigated after parenteral dosing of radiolabeled TAK-448 ([d-Tyr-¹⁴C]TAK-448) to rats and dogs, and it was confirmed if the radiolabeling position at d-Tyr was eligible for assessment of in vivo disposition.

2. Dosed radioactivity was rapidly and well absorbed after subcutaneous administration and an appreciable amount of unchanged TAK-448 (TAK-448F) and a hydrolyzed metabolite, M-I, were detected in the plasma of rats and dogs.

3. After intravenous administration of [d-Tyr-¹⁴C]TAK-448 to rats, the radioactivity widely distributed to tissues with relatively higher concentrations in kidney and urinary bladder.

4. The radioactivity was decreased rapidly from the tissues.

5. After subcutaneous administration of [d-Tyr-¹⁴C]TAK-448 to rats and dogs, the dosed radioactivity was almost completely recovered by 48 and 72?h in rats and dogs, respectively, and most of the radioactivity was excreted in urine after extensive metabolism in the two species.

6. These results suggest that TAK-448 has an acceptable pharmacokinetic profile for clinical evaluation and development, and demonstrate that the synthesized [D-Tyr-¹⁴C]TAK-448 used in this study represents a favorable labeling position to evaluate disposition properties of this compound.

     

Effects of triptolide on pharmacokinetics of fenofibrate in rats and its potential mechanism

1. Triptolide and fenofibrate are often used together for the treatment of nephrotic syndrome in Chinese clinics.

2. This study investigates the effects of triptolide on the pharmacokinetics of fenofibrate in rats and it potential mechanism.

3. The pharmacokinetics of fenofibrate (20?mg/kg) with or without triptolide pretreatment (2?mg/kg/day for seven days) were investigated. Additionally, the inhibitory effects of triptolide on the metabolic stability of fenofibrate were investigated using rat liver microsome incubation systems.

4. The results indicated that the C_max (35.34?±?7.52 vs. 30.43?±?6.45?μg/mL), t_1/2 (6.17?±?1.15 vs. 4.90?±?0.82?h) and AUC_(0–t) (468.12?±?35.84 vs. 416.35?±?32.68?mg?h?L^?1) of fenofibric acid decreased significantly (p?<?.05). The T_max of fenofibric acid increased significantly (p?<?.05) from 5.12?±?0.36 to 6.07?±?0.68?h. Additionally, the metabolic stability of fenofibrate was prolonged from 35.8?±?6.2 to 48.6?±?7.5?min (p?<?.05) with the pretreatment of triptolide.

5. In conclusion, these results indicated that triptolide could affect the pharmacokinetics of fenofibric acid, possibly by inhibiting the metabolism of fenofibrate in rat liver when they were co-administered.

     

Effect of pretreatment regimens of 1-aminobenzotriazole on metabolism and gastric emptying of probe compounds in rat

1. 1-Aminobenzotriazole (ABT) is a mechanism-based inactivator of major cytochrome P450 (CYP) enzymes, which is used in multiple mechanistic studies.

2. The purpose was to evaluate the effect of 2 and 16-h pretreatment regimens of ABT on the exposures of triazolam in rat. Another objective was to evaluate the effect of ABT on gastric emptying of acetaminophen.

3. Plasma area under the curve (AUC) of triazolam was increased by 101-fold and 81-fold for the rats pretreated with ABT at 2 and 16?h, respectively, compared to control rats. Time to reach maximum concentration was 0.3, 4.8 and 3.7?h in control, 2 and 16-h pretreatment animals, respectively. In the case of acetaminophen, where T_max was not delayed, the mean absorption time (MAT) in control, 2 and 16?h ABT pretreatment groups were 0.3, 4.6 and 2.9?h, respectively, suggesting delayed absorption. This hypothesis was further supported by GastroPlus^TM simulation.

4. In summary, extent of triazolam absorption was increased to a similar extent with both 2 and 16?h ABT pretreatment regimens, suggesting that either of the regimen can be used to increase parent exposures in rat. With ABT pretreatment, delayed absorption of triazolam and acetaminophen was observed, as suggested by delay in T_max and MAT, respectively.

     

Preclinical assessment of the ADME,efficacy and drug-drug interaction potential of a novel NAMPT inhibitor

1. GNE-617 (N-(4-((3,5-difluorophenyl)sulfonyl)benzyl)imidazo[1,2-a]pyridine-6-carboxamide) is a potent, selective nicotinamide phosphoribosyltransferase (NAMPT) inhibitor being explored as a potential treatment for human cancers.

2. Plasma clearance was low in monkeys and dogs (9.14?mL min^?1?kg^?1 and 4.62?mL min^?1?kg^?1, respectively) and moderate in mice and rats (36.4?mL min^?1?kg^?1 and 19.3?mL min^?1?kg^?1, respectively). Oral bioavailability in mice, rats, monkeys and dogs was 29.7, 33.9, 29.4 and 65.2%, respectively.

3. Allometric scaling predicted a low clearance of 3.3?mL min^?1?kg^?1 and a volume of distribution of 1.3?L kg^?1 in human.

4. Efficacy (57% tumor growth inhibition) in Colo-205 CRC tumor xenograft mice was observed at an oral dose of 15?mg/kg BID (AUC?=?10.4?µM h).

5. Plasma protein binding was moderately high. GNE-617 was stable to moderately stable in vitro. Main human metabolites identified in human hepatocytes were formed primarily by CYP3A4/5. Transporter studies suggested that GNE-617 is likely a substrate for MDR1 but not for BCRP.

6. Simcyp^® simulations suggested a low (CYP2C9 and CYP2C8) or moderate (CYP3A4/5) potential for drug-drug interactions. The potential for autoinhibition was low.

7. Overall, GNE-617 exhibited acceptable preclinical properties and projected human PK and dose estimates.

     

Model-based pharmacokinetic and pharmacodynamic analysis for acute effects of a small molecule inhibitor of diacylglycerol acyltransferase-1 in the TallyHo/JngJ polygenic mouse

1. The purpose of this study was to evaluate the acute effect of a small molecule inhibitor of DGAT-1 on triglycerides (TG) and cholesterol in polygenic type 2 diabetic TallyHo/JngJ (TH) mice. PF-04620110, a potent and selective DGAT-1 inhibitor, was used as a model compound in this study and which was administered to TH and ICR mice.

2. The concentration of the model compound that produced 50% of maximum lowering of TG level (IC₅₀) in TH mice was not significantly different from that in ICR mice, when estimated using the model-based pharmacokinetic and pharmacodynamic assay, a two-compartmental model and an indirect response model.

3. The clearance of the inhibitor in TH mice was fivefold higher than that in ICR mice, suggesting significantly altered pharmacokinetics. Moreover, the in vitro metabolic elimination kinetic parameters (k_e,met), determined using liver microsomes from TH and ICR mice were 1.24?±?0.14 and 0.174?±?0.116?min^?1, respectively.

4. Thus, we report that the differences in the acute effects of the small molecule DAGT-1 inhibitor between TH mice and ICR mice can be attributed to altered pharmacokinetics caused by an altered metabolic rate for the compound in TH mice.

     

Effects of CYP3A4*1G and CYP3A5*3 polymorphisms on pharmacokinetics of tylerdipine hydrochloride in healthy Chinese subjects

1. The aim of this analysis was to explore the influence of CYP3A4*1G and CYP3A5*3 polymorphisms on the pharmacokinetics of tylerdipine in healthy Chinese subjects.

2. A total of 64 and 63 healthy Chinese subjects were included and identified as the genotypes of CYP3A4*1G and CYP3A5*3, respectively. Plasma samples were collected for up to 120?h post-dose to characterize the pharmacokinetic profile following single oral dose of the drug (5, 15, 20, 25 and 30?mg). Plasma levels were measured by a high-performance liquid chromatography-mass spectrometry (LC-MS/MS). The pharmacokinetic parameters were calculated using non-compartmental method. The maximum concentration (C_max) and the area under the curve (AUC_0–24?h) were all corrected by the dose given.

3. In the wild-type group, the mean dose-corrected AUC_0–24?h was 1.35-fold larger than in CYP3A4*1G carriers (p?=?.018). Among the three CYP3A5 genotypes, there showed significantly difference (p?=?.008) in the t_1/2, but no significant difference was observed for the AUC_0–24?h and C_max. In subjects with the CYP3A5*3/*3 genotype, the mean t_1/2 was 1.35-fold higher than in CYP3A5*1/*1 group (p?=?.007). And the t_1/2 in CYP3A5*3 carriers also was 1.32-fold higher than in the wild-type group (p?=?.004).

4. CYP3A4*1G and CYP3A5*3 polymorphisms may influence tylerdipine pharmacokinetic in healthy Chinese subjects.

     

In vitro and in vivo pharmacokinetic characterization of mavacamten,a first-in-class small molecule allosteric modulator of beta cardiac myosin

1. Mavacamten is a small molecule modulator of cardiac myosin designed as an orally administered drug for the treatment of patients with hypertrophic cardiomyopathy. The current study objectives were to assess the preclinical pharmacokinetics of mavacamten for the prediction of human dosing and to establish the potential need for clinical pharmacokinetic studies characterizing drug–drug interaction potential.

2. Mavacamten does not inhibit CYP enzymes, but at high concentrations relative to anticipated therapeutic concentrations induces CYP2B6 and CYP3A4 enzymes in vitro. Mavacamten showed high permeability and low efflux transport across Caco-2 cell membranes. In human hepatocytes, mavacamten was not a substrate for drug transporters OATP, OCT and NTCP. Mavacamten was determined to have minimal drug–drug interaction risk.

3. In vitro mavacamten metabolite profiles included phase I- and phase II-mediated metabolism cross-species. Major pathways included aromatic hydroxylation (M1), aliphatic hydroxylation (M2); N-dealkylation (M6), and glucuronidation of the M1-metabolite (M4). Reaction phenotyping revealed CYPs 2C19 and 3A4/3A5 predominating.

4. Mavacamten demonstrated low clearance, high volume of distribution, long terminal elimination half-life and excellent oral bioavailability cross-species.

5. Simple four-species allometric scaling led to predicted plasma clearance, volume of distribution and half-life of 0.51?mL/min/kg, 9.5?L/kg and 9?days, respectively, in human.

     

Identification of UGTs and BCRP as potential pharmacokinetic determinants of the natural flavonoid alpinetin

1. Alpinetin is a natural flavonoid showing a variety of pharmacological effects such as anti-inflammatory, anti-tumor and hypolipidemic activities. Here, we aim to determine the roles of UDP-glucuronosyltransferases (UGTs) and breast cancer resistance protein (BCRP) in disposition of alpinetin.

2. Glucuronidation potential of alpinetin was evaluated using pooled human liver microsomes (pHLM), pooled human intestine microsomes (pHIM) and expressed UGT enzymes supplemented with the cofactor UDPGA. Activity correlation analyses with a bank of individual HLMs were performed to identify the main contributing UGT isozymes in hepatic glucuronidation of alpinetin. The effect of BCRP on alpinetin disposition was assessed using HeLa cells overexpressing UGT1A1 (HeLa1A1) cells.

3. Alpinetin underwent extensive glucuronidation in pHLM and pHIM, generating one glucuronide metabolite. Of 12 test UGT enzymes, UGT1A3 was the most active one toward alpinetin with an intrinsic clearance (CL_int?=?V_max/K_m) value of 66.5?μl/min/nmol, followed by UGT1A1 (CL_int?=?48.6?μl/min/nmol), UGT1A9 (CL_int?=?21.0?μl/min/nmol), UGT2B15 (CL_int?=?16.7?μl/min/nmol) and UGT1A10 (CL_int?=?1.60?μl/min/nmol). Glucuronidation of alpinetin was significantly correlated with glucuronidation of estradiol (an activity marker of UGT1A1), chenodeoxycholic acid (an activity marker of UGT1A3), propofol (an activity marker of UGT1A9) and 5-hydroxyrofecoxib (an activity marker of UGT2B15), confirming the important roles of UGT1A1, UGT1A3, UGT1A9 and UGT2B15 in alpinetin glucuronidation. Inhibition of BCRP by its specific inhibitor Ko143 significantly reduced excretion of alpinetin glucuronide, leading to a significant decrease in cellular glucuronidation of alpinetin.

4. Our data suggest UGTs and BCRP as two important determinants of alpinetin pharmacokinetics.

     

Impact of collagen-induced arthritis on the pharmacokinetic disposition of voriconazole,a widely used antifungal agent: in vitro and in vivo investigations in DBA/1J mice

1. Pharmacokinetics of voriconazole, an anti-fungal agent, was determined in collagen-induced arthritic (CIA) and healthy DBA/1J mice. CIA was confirmed in DBA/1J mice by clinical scoring and histological analysis.

2. In vivo oral pharmacokinetic study (3?mg/kg) and in vitro stability assessment in liver microsomes were performed in CIA vs. healthy DBA/1J mice. Additionally, hepatic portal vein cannulated (HPVC) CIA and healthy mice were used to clarify the role of gut first-pass effect. Voriconazole/N-oxide metabolite was measured in plasma and in vitro samples using liquid chromatography tandem-mass spectrometry method.

3. Voriconazole exposure was reduced in CIA by 27% as compared to healthy mice. Formation of voriconazole N-oxide was higher in CIA mice as evidenced by higher molar C_max ratio (i.e. metabolite/parent) of 2.08 vs. 1.66 in healthy mice. Because voriconazole was stable in microsomes, involvement of presystemic gut metabolism was suspected for decreased voriconazole exposure and formation of higher molar ratio of metabolite. HPVC work revealed higher formation of voriconazole N-oxide in CIA relative to healthy mice resulting in C_max/AUC ratios of 0.41/0.54 and 0.08/0.17, respectively, confirming first-pass effect.

4. The findings may have implications in the clinical therapy of arthritis patients who are concomitantly given voriconazole for the management of fungal infections.

     

Pharmacokinetics of anticoagulants apixaban,dabigatran, edoxaban and rivaroxaban in elderly Japanese patients with atrial fibrillation treated in one general hospital

1. Steady-state plasma concentrations of anticoagulants and the time since the previous administration in mainly outpatients with atrial fibrillation administered standard or reduced doses were analyzed for 110 elderly Japanese subjects (mean age, 76?years) treated with apixaban (2.5 or 5.0?mg twice daily), dabigatran etexilate (110 or 150?mg twice daily), edoxaban (30 or 60?mg once daily) or rivaroxaban (10 or 15?mg once daily) at one general hospital.

2. The pharmacokinetics in patients treated with standard and reduced doses of the four anticoagulants using liquid chromatography-tandem mass spectrometry was compared with the concentration ranges estimated using physiologically based pharmacokinetic modeling. Reduced doses of anticoagulants resulted in relatively small pharmacokinetic variations compared with the standard dose.

3. Statistical analyses revealed that renal impairment is likely not the sole determinant factor for high plasma concentrations of apixaban, dabigatran, edoxaban and rivaroxaban.

4. Patients with atrial fibrillation should be treated with the correct doses of oral anticoagulants as specified in the package inserts (e.g. reduced doses for elderly patients, patients with low body weights and in combination with P-glycoprotein inhibitor drugs) to avoid excessive or insufficient doses of direct oral anticoagulants.

     

Prediction of human pharmacokinetics of typical compounds by a physiologically based method using chimeric mice with humanized liver

1. In this study, total body clearance (CL_t), volume of distribution at steady state (V_ss) and plasma concentration–time profiles in humans of model compounds were predicted using chimeric mice with humanized livers.

2. On the basis of assumption that unbound intrinsic clearance (CL_Uint) per liver weight in chimeric mice was equal to those in humans, CL_t were predicted by substituting human liver blood flow and liver weights in well-stirred model. V_ss were predicted by Rodgers equation using scaling factors of tissue-plasma concentration ratios (SF_Kp) in chimeric mice estimated from a difference between the observed and predicted V_ss. These physiological approaches showed high prediction accuracy for CL_t and V_ss values in humans.

3. We compared the predictability of CL_t and V_ss determined by the physiologically based predictive approach using chimeric mice with those from predictive methods reported by Pharmaceutical Research Manufacturers of America. The physiological approach using chimeric mice indicated the best prediction accuracy in each predictive method.

4. Simulation of human plasma concentration–time profiles were generally successful with physiologically based pharmacokinetic (PBPK) model incorporating CL_Uint and SF_Kp obtained from chimeric mice.

5. Combined application of chimeric mice and PBPK modeling is effective for prediction of human PK in various compounds.

     

Pharmacokinetics and bioequivalence of two strontium ranelate formulations after single oral administration in healthy Chinese subjects

1. Pharmacokinetics of exogenous strontium (Sr) and bioequivalence of a new oral formulation of strontium ranelate compared with the brand-name drug in healthy Chinese subjects was evaluated.

2. A balanced, randomized, single-dose, two-treatment parallel study was conducted in 36 healthy Chinese subjects. Subjects were randomly allocated into two groups of 18 to receive a single oral dose of test formulation and reference formulation under a fasting state, respectively. Blood samples were collected at 19 designated time points up to 240-h post-dose. Serum concentrations of Sr were quantified by ICP-MS.

3. A total of 36 subjects were enrolled and completed the study. Nine mild adverse events in 6 subjects were reported. The C_max, AUC_0–72?h, AUC_0–t, and AUC_0–∞ of test and reference formulations shown as mean?±?SD were 6.97?±?1.78 and 6.78?±?1.80?µg/mL, 199?±?51 and 187?±?38?µg·h/mL, 303?±?89 and 278?±?54?µg·h/mL, and 337?±?109 and 305?±?60?µg·h/mL, respectively.

4. Two formulations were bioequivalent, and both were generally well tolerated.

     

Functional assessment of rat pulmonary flavin-containing monooxygenase activity

1. The expression of flavin-containing monooxygenase (FMO) varies extensively between human and commonly used preclinical species such as rat and mouse. The aim of this study was to investigate the pulmonary FMO activity in rat using benzydamine. Furthermore, the contribution of rat lung to the clearance of benzydamine was investigated using an in vivo pulmonary extraction model.

2. Benzydamine N-oxygenation was observed in lung microsomes and lung slices. Thermal inactivation of FMO and CYP inhibition suggested that rat pulmonary N-oxygenation is predominantly FMO mediated while any contribution from CYPs is negligible.

3. The predicted lung clearance (CL_lung) estimated from microsomes and slices was 16?±?0.6 and 2.1?±?0.3?mL/min/kg, respectively. The results from in vivo pulmonary extraction indicated no pulmonary extraction following intravenous and intra-arterial dosing to rats. Interestingly, the predicted CL_lung using rat lung microsomes corresponded to approximately 35% of rat CL_liver suggesting that the lung makes a smaller contribution to the whole body clearance of benzydamine.

4. Although benzydamine clearance in rat appears to be predominantly mediated by hepatic metabolism, the data suggest that the lung may also make a smaller contribution to its whole body clearance.

     

Development of simultaneous quantification method of loteprednol etabonate (LE) and its acidic metabolites,and analysis of LE metabolism in rat

1. Loteprednol etabonate (LE) is a soft corticosteroid with two labile ester bonds at 17α- and 17β-positions. Its corticosteroidal activity disappears upon hydrolysis of either ester bond. Hydrolysis of both ester bonds produces the inactive metabolite, Δ¹-cortienic acid (Δ¹-CA).

2. The simple high-performance liquid chromatography method using acetic acid gradient was developed for the simultaneous determination of LE and its acidic metabolites.

3. LE was hydrolyzed in rat plasma with a half-life of 9?min. However, LE hydrolysis was undetectable in rat liver and intestine. LE hydrolysis in rat plasma was completely inhibited by paraoxon and bis(p-nitrophenyl) phosphate, thus identifying carboxylesterase as the LE hydrolase. Rat plasma carboxylesterase had a K_m of 6.7?μM for LE.

4. In contrast to the disappearance rate of LE in rat plasma, the formation rate of 17α-monoester and Δ¹-CA was markedly low, and a main hydrolysate of LE was not detected in rat plasma.

5. The metabolism of LE proceeded via different pathways in human and rat plasma. LE was slowly hydrolyzed by paraoxonase in human plasma to 17α-monoester with a half-life of 12?h, but by carboxylesterase in rat plasma to yield undetectable products, presumed to include the unstable 17β-monoester.