Relative contribution of the gut, liver, and lung to the first-pass hydrolysis (bioactivation) of orally administered 14C-fosinopril sodium in dogs. In vivo and in vitro studies

The relative contribution of the gut, liver, and lung to the first-pass hydrolysis (bioactivation) of the orally administered prodrug, fosinopril sodium (FS), to the active angiotensin-converting enzyme (ACE) inhibitor, SQ 27,519 (S), was determined. Two dogs each received 14C-FS by the following routes of administration: oral, intraportal, and intra-arterial. Extraction ratios (E) for the gut and liver were calculated based on the relative ratios of the AUC of FS in arterial plasma after administration of FS by various routes. The high intrinsic capability of the gut and liver to hydrolyze FS was reflected by E values which ranged from 69 to 91%. Since the gut is the first site after an oral dose, its contribution to the overall first-pass hydrolysis (greater than 75% of the absorbed dose) was estimated to be significantly greater than that of the liver (less than 25% of the absorbed dose). Concentrations of FS were similar in central arterial and venous plasma after a steady state arterial infusion of 14C-FS, indicating that the lung is apparently not a site of prodrug hydrolysis. This conclusion was consistent with the results of in vitro studies that indicated the following order of esterase activity: liver = kidney much greater than small intestine greater than blood, aorta, and lung. When data from in vitro studies were extrapolated to the in vivo situation, the blood itself was not a significant site for hydrolysis of FS in dogs. Based on the body clearance of FS (approximately 30 ml/min/kg) estimated after the intra-arterial route, roughly 50% of the systemic hydrolysis of the prodrug appears to occur at extrahepatic site(s), such as the kidney.     

Potential Improvement in Shelf Life Using the Prodrug Approach. II. A Systematic Examination of Kinetic Requirements

The utilization time (UT) for a solution of a prodrug that is rapidly and completely converted to drug in the blood may be longer than the time for 10% loss of the initial concentration. The UT for an intravenous prodrug solution is the period during which the total prodrug and drug concentration exceeds 90% of the initial concentration. The influence of the rate of prodrug degradation (k _nc), its conversion (k _c) to drug, and the subsequent drug degradation (k _h) on the UT of a stored solution was examined by simulating the prodrug and drug concentration–time courses. The ratio of the shelf life of a prodrug solution to that of the parent drug (UT_ratio) was calculated using a wide range of values for the three rate constants. Three-dimensional plots relating the UT_ratio to the k _c, k _nc, and k _h values provide a basis for making a priori assessments of kinetic requirements for designing a prodrug to increase storage time. A parenteral prodrug intended to increase storage time may have a larger overall rate of loss than the parent drug, but it must have a smaller degradation rate (k _nc < k _h) to be successful. The UT for an oral prodrug solution depends upon the bioavailability of the prodrug relative to the drug in addition to the values for k_nc, k _c, and k _h. Two ampicillin prodrugs were used as models to calculate actual UT_ratio versus pH profiles. Intravenous solutions showed modest gains in the UT_ratio in the acid region, whereas oral solutions reached a UT_ratio as high as 22 by combining favorable rate constants with increased bioavailability. These actual UT_ratio versus pH profiles were interpreted in terms of the theory established using the simulations.     

Preclinical pharmacology of zofenopril, an inhibitor of angiotensin I converting enzyme

Zofenopril calcium (one-half calcium salt) is a prodrug ester analog of captopril whose biological effects are manifested by its active component, SQ 26,333. Because of the relative insolubilities of both zofenopril calcium and SQ 26,333, zofenopril potassium salt and SQ 26,703, the arginine salt of the active ACE (angiotensin I converting enzyme) inhibitory moiety of zofenopril, were employed in many of the following studies. The in vitro and in vivo pharmacological effects of zofenopril have been evaluated and comparisons have been made to captopril. In vitro, SQ 26,703 was more potent than captopril as an inhibitor of rabbit lung ACE (IC50 = 8 vs. 23 nM). SQ 26,703 was also a potent inhibitor of angiotensin I (AI)-induced contractions (EC50 = 3 nM) and a potentiator of bradykinin-induced contractions (EC50 = 1 nM) of isolated guinea pig ileum, while it had no effect on the inotropic effects of angiotensin II, BaCl2, PGE1, histamine, serotonin, or acetycholine in the same tissue, signifying that zofenopril is a specific inhibitor of ACE. In vivo, the potency of SQ 26,703 was equal to or greater than that of captopril as an inhibitor of an AI pressor response when given intravenously to rats, dogs, and monkeys. After oral administration of equimolar doses, zofenopril was the more effective and longer lasting ACE inhibitor in all three species. In SHR, doses of 6.6 and 22.0 mg/kg, p.o. lowered pressure by 20 and 33 mm Hg, respectively, while 30 mg/kg of captopril lowered pressure by 25 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of the intrinsic clearance of donepezil (Aricept®) between in vivo and in vitro studies in rat,dog and human

1. Donepezil hydrochloride (Aricept®) is used for the treatment of Alzheimer's disease. Here the correlation of the intrinsic clearance (Cl_int) of donepezil between the in vivo and in vitro states was studied in rat, dog and human. 2. In an experiment with ¹⁴C-donepezil and human microsomes the routes of metabolism were identified as N-dealkylation and O-demethylation, and no unknown metabolites were detected. 3. The Cl_int of donepezil in the male rat, female rat, dog and human liver microsomes were 33.7, 13.4, 37.0 and 6.35 μl/min/mg microsomal protein respectively, and sex difference in rat and interspecies difference in the estimated Cl_int were found. 4. After a single intravenous administration to the male rat, female rat and dog, total plasma clearance (Cl^P_total) was 78.6, 29.5 and 88.3 ml/min/kg respectively, and a sex difference was observed in rat. 5. After a single oral administration to the male rat, dog and healthy volunteer, Cl^P_total/F was 140, 105 and 2.35 ml/min/kg respectively, and remarkable differences were observed between animals and man. 6. The contribution of renal clearance to blood clearance (Cl_r) was low in all species. The predicted in vitro hepatic clearance (Cl_h-pre) was in the rank order: male rat (15.91 ml/min/kg) &gt; dog (7.96) &gt; female rat (7.67) &gt; human (1.04). Although Cl_h-pre was underestimated, Cl_h-pre was significantly correlated with that of ClBtotal in the different animal species and in man, indicating that the in vitro-in vivo ranking order was conserved.     

Utility of Göttingen minipigs for the prediction of human pharmacokinetic profiles after intravenous drug administration

Göttingen minipigs are increasingly used to evaluate the pharmacokinetic (PK) profiles of drug candidates. However, their accuracy in predicting human PK parameters is unclear. In this study, we investigated the utility of Göttingen minipigs for predicting human PK profiles. We evaluated the PK parameters of 30 compounds with diverse metabolic pathways after intravenous administration in minipigs. Human total clearance (CL_total) was corrected using the blood to plasma ratio, and the volume of distribution at steady state (Vd_(ss)) was corrected with plasma unbound fraction (fup). CL_total and Vd_(ss) were predicted using single-species allometric scaling using data from minipigs and other reported animal models (monkeys, human liver chimeric mice, and rats). The predicted values were compared with actual values reported in humans. Göttingen minipig were superior to rats because of their better predictability of Vd_(ss) and CL_total, as represented by lower absolute average fold error values. However, their predictability for Vd_(ss) was inferior to monkey and human liver chimeric mice. Prediction of CL_total from blood-based minipig data showed excellent correlation with human data, and comparable predictability with monkey and human liver chimeric mice. Thus, Göttingen minipigs can be used as an optional model for preclinical pharmaceutical research for predicting human CL_total.     

Potential Improvement in the Shelf Life of Parenterals Using the Prodrug Approach: Bacampicillin and Talampicillin Hydrolysis Kinetics and Utilization Time

The utilization time for a parenteral prodrug solution with a bioavailable fraction of unity was defined as the time during which the total of the prodrug concentration and the drug concentration equals or exceeds 90% of the initial prodrug concentration. This utilization time was calculated as a function of pH, buffer, and temperature using the experimentally determined rate expressions for bacampicillin and talampicillin. The results were compared to the shelf life of ampicillin solutions under identical storage conditions. First-order rate constants were determined for conversion of the prodrugs to ampicillin (k _c), for -lactam degradation of the prodrugs (k _nc), for the overall loss of prodrugs (k _sum), and for -lactam degradation of ampicillin (k _h) in aqueous solutions at 25.0 to 60.0°C, µ = 0.5, in the pH range 0.90 to 8.4. Loss of bacampicillin proceeded primarily by degradation at pH levels below 4 but was due predominantly to conversion at pH levels above 5. Loss of talampicillin was due primarily to conversion throughout the entire pH range. While the prodrug utilization times were approximately twice the shelf life of ampicillin in acidic solutions, ampicillin was significantly better in neutral solutions. The results illustrate the potential for increased prodrug storage periods when utilization time is defined on the basis of the bioactivity rather than on the prodrug concentration alone.     

Uptake–toxicity relationships of a series of N-substituted N′-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices

A previous study showed that the cytotoxicity of a series of N-p-phenyl-substituted N′-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices increased with increasing electron-withdrawing capacity of the p-substituent and may be related to the V_max/K_m values of bioactivation of the thiourea-moiety by hepatic flavin-containing monooxygenases (FMOs). However, differences in the uptake of xenobiotics into precision-cut liver slices can also have consequences for the rates of metabolism of xenobiotics. In the present study, therefore, we investigated the rate and nature of uptake of 9 N-substituted N′-(4-imidazole-ethyl)thiourea into precision-cut rat liver slices. It was found that a five-fold difference exists among a series of N-substituted N′-(4-imidazole-ethyl)thiourea both in the initial rate of uptake and in the steady-state levels ultimately achieved in the precision-cut rat liver slices. It appeared that the most cytotoxic compounds were also the most readily absorbed compounds. The concentration-dependent initial rate of uptake could be described by a carrier-mediated saturable component and a non-saturable component. At cytotoxic concentrations, the non-saturable component accounted for more than 95% of the total uptake. From this study, it is concluded that differences in rate of uptake of thiourea-containing compounds may be a contributing factor to the differences in bioactivation by FMOs as the basis of the structure–toxicity relationships observed in precision-cut rat liver slices.     

Synthesis and Characterization of 9-[P-(N,N-dipropyl Sulfamide)] Benzoylamino-1,2,3,4-4H-acridine—A Potential Prodrug for the CNS Delivery of Tacrine

9-[P-(N,N-dipropylsulfamide)]benzoylamino-1,2,3,4-4H-acridine (PTHA) was synthesized as a prodrug to improve the curative effect of tacrine hydrochloride (THA), to prolong the activation time in brain, and to decrease the hepatic toxicity. The prodrug was prepared by attaching carboxyl group of probenecid to 9-amino group of THA. The structure of PTHA was confirmed by IR, ₁HNMR, MS and UV. The physiochemical properties and stabilities of the prodrug under various conditions were investigated. Possibility of the prodrug passing through the blood–brain barrier (BBB) was evaluated in vitro by biopartition micellar chromatography (BMC). The concentrations of THA and PTHA in various tissues were determined by reversed-phase high-performance liquid chromatography after intravenous (i.v.) administration. The results showed PTHA was stable in various pHs and temperatures. It was indicated by partition coefficient in ethyl acetate/water that lipophilicity of the prodrug increased and it was predicted by BMC that the prodrug could improve the infiltration ability across BBB of THA. In-vivo experiment showed the concentrations of THA in brain and liver kept stable for about 4 h, which was beneficial for the treatment of Alzheimer's disease (AD). Compared with THA at the same time, AUC in liver decreases significantly, the overall targeting efficiency (TE) was enhanced from 10.97 to 16.11% and AUC_brain/AUC_liver increased from 1.52 to 2.53, which suggests the possibility to reduce the hepatic toxicity of THA by the way of the prodrug.     

CBR1 rs9024 genotype status impacts the bioactivation of loxoprofen in human liver

Loxoprofen is an anti‐inflammatory drug that requires bioactivation into the trans‐OH metabolite to exert pharmacological activity. Evidence suggests that carbonyl reductase 1 (CBR1) is important during the bioactivation of loxoprofen. This study examined the impact of the functional single nucleotide polymorphism CBR1 rs9024 on the bioactivation of loxoprofen in a collection of human liver samples. The synthesis ratios of trans‐OH loxoprofen/cis‐OH loxoprofen were 33% higher in liver cytosols from donors homozygous for the CBR1 rs9024 G allele in comparison with the ratios in samples from donors with heterozygous GA genotypes. Complementary studies examined the impact of CBR1 rs9024 on the bioactivation of loxoprofen in lymphoblastoid cell lines. CBR1 rs9024 genotype status impacts the synthesis of the bioactive trans‐OH metabolite of loxoprofen in human liver.     

Reversible metabolism of vitamin K-vitamin K epoxide: Modeling considerations and limitations

Due to the cyclical natural of the vitamin K-vitamin K epoxide system, a two-compartment reversible metabolism model was used to describe this interconversion. In attempting to apply this model to the vitamin K-vitamin K epoxide cycle using literature data from dogs, interconversion and elimination clearances were obtained which are not physiologic. Consequently, the assumptions of the model were reexamined with respect to their validity. One critical assumption of the twocompartment model for interconversion is that it can only be applied in the absence of flow limitations. To determine what effect flow limitations may exert on the vitamin K and vitamin K epoxide apparent blood clearances, a model separating the liver from the blood compartment was proposed assuming the interconversion and metabolism of vitamin K and its epoxide occurred only within the liver. Simulated data suggested that if the reversible metabolic clearance values exceeded the distributional clearance terms, all the apparent clearances calculated using blood concentration-time data were in error. It is suggested that a two-compartment interconversion model might be too simplistic for the vitamin K-vitamin K epoxide cycle where the reversible metabolism is efficient and the distributional clearance may be rate limiting.Glossary of symbols Dose ^E Dose of vitamin K epoxide - Dose ^K Dose of vitamin K AUCs calculated from blood concentration-time profile AUC _E ^E Area under curve for vitamin K epoxide after epoxide dose - AUC _E ^K Area under curve for vitamin K after epoxide dose - AUC _K ^K Area under curve for vitamin K after vitamin K dose - AUC _E ^K Area under curve for vitamin K epoxide after vitamin K dose AUCs calculated from liver concentration-time profile AUC _L,E ^E Area under curve for vitamin K epoxide after epoxide dose - AUC _L,K ^E Area under curve for vitamin K after epoxide dose - AUC _L,K ^K Area under curve for vitamin K after vitamin K dose - AUC _L,E ^K Area under curve for vitamin K epoxide after vitamin K dose Clearances calculated from blood AUCs Cl _E ^B Elimination clearance of vitamin K epoxide - Cl _K ^B Elimination clearance of vitamin K - Cl _EK ^B Conversion clearance from vitamin K epoxide to vitamin K - Cl _KE ^B Conversion clearance from vitamin K to vitamin K epoxide Clearances calculated from liver AUCs Cl _E ^L Elimination clearance of vitamin K epoxide - Cl _K ^L Elimination clearance of vitamin K - Cl _EK ^L Conversion clearance from vitamin K epoxide to vitamin K - Cl _KE ^L Conversion clearance from vitamin K to vitamin K epoxide Distributional clearances Cl _K ^BL Clearance of vitamin K from the blood to the liver - Cl _K ^LB Clearance of vitamin K from the liver to the blood - Cl _E ^BL Clearance of vitamin K epoxide from the blood to the liver - Cl _E ^LB Clearance of vitamin K epoxide from the liver to the blood Supported by a grant from The American Heart Association, Kentucky Affiliate.     

Relative Lipophilicities and Structural-Pharmacological Considerations of Various Angiotensin-Converting Enzyme (ACE) Inhibitors

Lipophilicities of seven structurally diverse angiotensin-converting enzyme (ACE) inhibitors, viz., captopril, zofenoprilat, enalaprilat, ramiprilat, lisinopril, fosinoprilat, and ceronapril (SQ29852), were compared by determining their octanol-water distribution coefficients (D) under physiological pH conditions. The distribution coefficients of zofenopril, enalapril, ramipril and fosinopril, which are the prodrug forms of zofenoprilat, enalaprilat, ramiprilat, and fosinoprilat, respectively, were also determined. Attempts were made to correlate lipophilicities with the reported data for oral absorption, protein binding, ACE inhibitory activity, propensity for biliary excretion, and penetration across the blood-brain barrier for these therapeutic entities. Better absorption of prodrugs compared to their respective active forms is in agreement with their greater lipophilicities. Captopril, lisinopril, and ceronapril are orally well absorbed despite their low lipophilicities, suggesting involvement of other factors such as a carrier-mediated transport process. Of all the compounds studied, the two most lipophilic ACE inhibitors, fosinoprilat and zofenoprilat, exhibit a rank-order correlation with respect to biliary excretion. This may explain the dual routes of elimination (renal and hepatic) observed with fosinoprilat in humans. The more lipophilic compounds also exhibit higher protein binding. Both the lipophilicity and a carrier-mediated process may be involved in penetration of some of these drugs into brain. For structurally similar compounds, in vitro ACE inhibitory activity increased with the increase in lipophilicity. However, no clear correlation between lipophilicity and ACE inhibitory activity emerged when different types of inhibitors are compared, possibly because their interactions with enzymes are primarily ionic in nature.     

Effect of serum protein binding on pharmacokinetics and anticoagulant activity of phenprocoumon in rats

The relationship among serum protein binding, kinetics of elimination, distribution, and anticoagulant activity of phenprocoumon was investigated in 25 selected outbred Sprague-Dawley rats which differed in the extent of serum protein binding of this drug. In addition, the serum protein binding of phenprocoumon was altered in inbred Lewis rats by continuous treatment with tolbutamide. This drug was found to displace phenprocoumon from serum proteins without affecting its intrinsic clearance. The serum free fraction values (f_s)of the selected Sprague-Dawley rats ranged from 0.0053 to 0.0145. There were positive and linear correlations between f_s and the first-order elimination rate constant (k), f_s and total clearance (CL _total ),and f_s and the liver/plasma concentration ratio (L/P ratio) of phenprocoumon. The free fraction values in the liver tissue (f _I )showed twofold variations and were not related to f_s.The half-effective plasma concentrations (C _p50% )of total phenprocoumon (i.e., the concentrations necessary to inhibit the prothrombin complex synthesis rate by 50%) decreased with increasing f_s.The C_p50% values of total drug varied eightfold between the animals but those of free drug only 3.5- fold. The total anticoagulant effect per dose (AE/dose), as reflected by the magnitude of the area above the prothrombin complex activity vs. time curve in the plasma, varied only 1.5- fold between the rats and was not related to f_s.Continuous treatment of inbred Lewis rats with tolbutamide led to an increase of f_s (twofold), k (1.3-fold), V_d (1.5-fold), and CL_total (twofold). The intrinsic clearance (CL _intr )remained unaffected. There was no significant increase of f_L but a twofold increase of the L/P ratio. AE/dose and the C_p50% values of free drug in tolbutamide-treated rats were not significantly different from those of control rats. Thus an increase of the free fraction of phenprocoumon in the serum of rats is followed by a proportional increase of the total clearance. This prevents a concomitant rise of the free drug concentration. Consequently, the total anticoagulant effect per dose remains almost unaffected by about threefold variations in the serum free fraction values of this drug.This work was supported by the Deutsche Forschungsgemeinschaft: it is part of the Ph.D. thesis for D. T.     

Pharmacokinetic Assessment of the Sites of First-pass Metabolism of BMS-181101, an Antidepressant Agent,in Rats

The relative contribution of the gut and the liver to the first-pass metabolism of BMS-181101 (3-[3-[4-(5-methoxy-4-pyrimidinyl)-1-piperazinyl]propyl]-5-fluoro-1H-indole dihydrochloride), a potential antidepressant agent, has been evaluated in rats. Nine male Sprague–Dawley rats were divided into three groups of three and each rat received a single 20 mg kg^?1 dose of [¹⁴C]BMS-181101 via a 30 min constant-rate intravenous infusion, a 30-min constant-rate intraportal infusion or oral gavage. Serial blood samples were collected for 8 h after dosing and plasma was analysed for unchanged BMS-181101 and total radioactivity. Extraction ratios for BMS-181101 by the gut and liver were calculated on the basis of ratios of the area under the plasma BMS-181101 concentration–time curve. The gut had a high intrinsic capacity for metabolizing BMS-181101-extraction ratios were 93% and 10% for the gut and liver, respectively. After oral administration BMS-181101 is sequentially exposed to the gut then the liver. As a result, the contribution of the gut to the overall first-pass effect (ca. 93%) was significantly greater than that of the liver     

肝靶向氟尿嘧啶类脂纳米粒的研究

目的　为了提高氟尿嘧啶(5-Fu)的疗效,降低其毒副作用,制备具肝靶向的5-Fu类脂纳米粒。方法　利用氟尿嘧啶与硬脂酰氯进行反应,制备了5-Fu前体药物N₁-硬脂酰-5-Fu,通过红外光谱及核磁共振谱对合成的目标化合物进行结构确认。同时研究了前体药物的性质及稳定性。采用物理凝聚法制备类脂纳米粒,并研究其形态、粒径及粒径分布、载药量、体外释药特征、动物体内分布与药代动力学参数等。结果　平均粒径d_av=240.19 nm,载药量为20.53%。体外释药速率符合一级动力学模型。与5-Fu水针剂比较,类脂纳米粒组在肝脏中药物含量平均增加了一倍以上。家兔体内主要药动学参数为：V_c=0.04336 L.kg^-1,T_1/2β=1.2834 h,CL=0.1632 L.h^-1。结论　利用前体药物可提高药物的脂溶性,首次以物理凝聚法制备类脂纳米粒,小鼠体内分布研究表明类脂纳米粒有明显的肝靶向,有一定的优越性和参考价值。     

Toxic effects of methylcyclopentadienyl manganese tricarbonyl (MMT) in rats: Role of metabolism

The toxicity of methylcyclopentadienyl manganese tricarbonyl (MMT) has been investigated in relation to its in vivo biotransformation in the rat. The LD₅₀ dose of MMT was found to be 50 mg/kg for oral administration and 23 mg/kg for ip administration. Death appeared to be caused by severe pulmonary hemorrhagic edema. Histological studies of MMT-treated animals revealed pathologic changes in lungs, liver, and kidney. Phenobarbital pretreatment protected rats from the lethal effects of 2.5 times the LD₅₀ dose of MMT, it shifted the site of tissue injury from the lungs to the liver, and it caused a doubling of the rate of biliary excretion of MMT metabolites. The possibility is discussed that MMT per se is toxic without bioactivation, and that the protective effect of phenobarbital pretreatment is due to a first-pass effect preventing toxic concentrations of orally administered MMT from reaching the systemic circulation.     

Rat cytochromes P450 (CYP) specifically contribute to the reductive bioactivation of AQ4N,an alkylaminoanthraquinone-di-N-oxide anticancer prodrug

1. The bioreductive activation of the alkylaminoanthraquinone di-N-oxide prodrug AQ4N has been characterized in rat hepatic tissue using HPLC. 2. AQ4N was shown to be metabolized to two products, namely AQM, the two electron reduced mono-N-oxide, and AQ4, the four electron reduced active cytotoxic agent. 3. Metabolism was shown to occur in microsomes with an apparent K_m=30.29 μM and V_max=1.05 nmol/mg/min. 4. Bioreduction was dependent on anaerobic conditions and the presence of the reduced cofactor NADPH. Ketoconazole (100 μM) and carbon monoxide both inhibited AQ4N metabolism inferring a role for cytochrome P450 (CYP). 5. Microsomes from phenobarbitone and isoniazid-pretreated animals significantly (p &lt; 0.05) enhanced the formation of AQ4 from AQ4N indicating a role for CYP2B and 2E respectively. The involvement of both CYP2B and 2E was confirmed by the use of CYP-specific inhibitors. 6. In conclusion, the involvement of rat hepatic CYP in the reductive bioactivation of the novel antitumour prodrug AQ4N has been established in detail for the first time. These findings highlight an important interspecies difference between the metabolism of AQ4N in rat and man which was shown earlier to be mediated by CYP3A enzymes. The pharmacological significance of this is discussed.     

Contribution of intestinal microfloral metabolism to the total macromolecular covalent binding of 1-nitro-pyrene in the lung and liver of the rat

1-Nitropyrene (NP) is a direct acting mutagen found in diesel exhaust and coal-combustion fly ash. The purpose of this study was to investigate the contribution of gut microfloral metabolism to the macromolecular covalent binding (MCB) of NP and/or its metabolites in lungs and liver of the rat. Normal and antibiotic treated rats were administered [14C]NP and MCB was quantitated at various times in lungs and livers. Abolition of gut microfloral metabolism by antibiotic treatment significantly altered total MCB in lungs. MCB in lungs of antibiotic-treated animals 4 h after oral administration of NP was 0.15 nmol NP equivalents/g and was significantly (P less than 0.05) decreased to less than one-half of control values (0.42 nmol NP equivalents/g). MCB in lungs of antibiotic-treated rats was no different from the controls 1 week after NP administration (0.1 nmol NP equivalents/g). Comparison of livers from control and antibiotic-treated rats demonstrated the same pattern of MCB as lungs but differences were not significant. These results reveal that metabolism by gut microflora may play a role in the activation and covalent binding of NP to macromolecules. However, the alteration of covalent binding observed after antibiotic treatment was a change in the time course of formation and breakdown of covalently bound forms and not an effect on the quantity of bound material remaining at 1 week indicating that gut microfloral metabolism is not an exclusive pathway for bioactivation of NP in the rat.     

Esterase-Sensitive Cyclic Prodrugs of Peptides: Evaluation of an Acyloxyalkoxy Promoiety in a Model Hexapeptide

Purpose. To evaluate a cyclic acyloxyalkoxycarbamate prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of the peptide and stabilize it to metabolism. Methods. Conversion to the linear hexapeptide was studied at 37°C in aqueous buffered solutions and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model. Results. In buffered solutions the cyclic prodrug degraded chemically to the linear hexapeptide in stoichiometric amounts. Maximum stability was observed between pH 3–4. In 90% human plasma (t _1/2 = 100 ± 4 min) and in homogenates of the rat intestinal mucosa (t _- = 136 ± 4 min) and rat liver (t _- = 65 ± 3 min), the cyclic prodrug disappeared faster than in buffered solution, pH 7.4 (t _- = 206 ± 11 min). Pretreatment of these media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug (t _- = 282 ± 25 min) was significantly more stable than the hexapeptide (t _- = 14 min) and at least 76-fold more able to permeate (P _app = 1.30 ± 0.15 × 10^–7 cm/ s) than the parent peptide (P _app 0.17 × 10^–8 cm/s). Conclusions. Preparation of a cyclic peptide using an acyloxyalkoxy promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.     

The combined impact of CYP2C19 and CYP2B6 pharmacogenetics on cyclophosphamide bioactivation

AIMS

The role of CYP pharmacogenetics in the bioactivation of cyclophosphamide is still controversial. Recent clinical studies have suggested a role for either CYP2C19 or CYP2B6. The aim of this study was to clarify the role of these pharmacogenes.

METHODS

We used a combined in vitro–in vivo approach to determine the role of these pharmacogenes in the bioactivation of the prodrug to 4-hydroxy cyclophosphamide (4-OHCP). Cyclophosphamide metabolism was determined in a human liver biobank (n = 14) and in patients receiving the drug for treatment of lupus nephritis (n = 16)

RESULTS

In livers of known CYP2C19 and CYP2B6 genotype and protein expression we observed that there was a combined role for both CYP2C19 and CYP2B6 in the bioactivation of cyclophosphamide in vitro. The presence of at least one loss of function (LoF) allele at either CYP2C19 or CYP2B6 resulted in a significant decrease in both V_max (P = 0.028) and CL_int (P = 0.0017) compared with livers with no LoF alleles. This dual genotype relationship was also observed in a preliminary clinical study, with patients who had ≥1 LoF allele at either CYP2C19 or CYP2B6 also displaying significantly (P = 0.0316) lower bioactivation of cyclophosphamide. The mean 4-OHCP : CP bioactivation ratio was 0.0014 (95% CI 0.0007, 0.002) compared with 0.0071 (95% CI 0.0001, 0.014) in patients with no LoF alleles at either of these genes.

CONCLUSIONS

The presence of ≥1 LoF allele(s) at either CYP2B6 or CYP2C19 appeared to result in decreased bioactivation of cyclophosphamide both in vitro and in patients. Further clinical studies to confirm this relationship are warranted.     

PEPT1-mediated prodrug strategy for oral delivery of peramivir

 Peramivir was a novel and highly potent neuraminidase (NA) inhibitor for the treatment of influenza A and B. However, it exhibited a very low oral bioavailability (only 3%) due to the high polarity (log P of ?1.4) and the low membrane permeability across the intestine. To utilize the PEPT1-mediated prodrug strategy to improve the oral absorption and develop the oral alternative, seven amino acid ester prodrugs and seven amino acid amide prodrugs have been synthesized. The permeability of these prodrugs across Caco-2 cells were screened. Peramivr-(CH2)2-l-Val and Peramivir-l-Ile were of the highest permeability in ester prodrugs and amide prodrugs, respectively, and then they were selected for further studies. Glycylsarcosine (gly-sar) uptake by Caco-2 could be inbihited by Peramivir-(CH2)2-l-Val and Peramivir-l-Ile in a concentration-dependent manner, and the IC50 was 1.34 ± 0.31 mM and 1.78 ± 0.48 mM, respectively. The direct uptake of Peramivir-(CH2)2-l-Val and Peramivirl-Ile in MDCK-PEPT1 cells were significantly higher than in MDCK mock cells, and could be markedly inhibited by gly-sar. The uptake of Peramivir-(CH2)2-l-Val and Peramivir-l-Ile (0.01 to 50 mM) in MDCK-hPEPT1 cells conformed to Michaelis–Menten Equation. The oral bioavailability of peramivir was 65.3% and 37.3% after the oral administration of Peramivir- (CH2)2-l-Val and Peramivir-l-Ile to rats, respectively. The oral absorption and bioactivation of Peramivir-(CH2)2-l-Val was rapid and extensive, and no Peramivir-(CH2)2-l-Val was found in plasma. Because the amide bond was relatively stable, Peramivir-l-Ile could not be totally converted to the parent drug in vivo. Peramivir-(CH2)2-l-Val with good oral profiles and rapid bioactivation might be a promising prodrug for the further clinic development. The present study also corroborated the idea that the PEPT1-mediated prodrug approach has enormous promise for improving the oral absorption of poorly absorbed drug.