Differences in background characteristics of patients with chronic hepatitis C who achieved sustained virologic response with interferon-free versus interferon-based therapy and the risk of developing hepatocellular carcinoma after eradication of hepatitis C virus in Japan.

We compared the background characteristics of patients with chronic hepatitis C who achieved eradication of hepatitis C virus (HCV), that is sustained virologic response (SVR), with interferon (IFN)-based versus IFN-free antiviral therapy in Japan. In addition, we used a previously reported risk assessment model to compare the incidence of hepatocellular carcinoma (HCC) after SVR by treatment type. Pretreatment characteristics of 1533 patients who achieved SVR with IFN-based therapy and 1086 patients with IFN-free therapy from five institutions across Japan were compared. The risk of HCC after SVR was assessed based on pretreatment characteristics, and the incidence of HCC after SVR was estimated in both groups. Age and serum alpha-fetoprotein levels were higher, platelet count was lower, and liver fibrosis was more advanced in patients who achieved SVR with IFN-free therapy compared with IFN-based therapy. The incidence of HCC after SVR in the IFN-free group was estimated to be more than twofold higher than in the IFN-based therapy group (7.29% vs. 3.09%, and 6.23% vs. 3.01% when excluding patients who have underwent curative treatment for HCC). There are large differences in pretreatment characteristics between patients who achieved SVR with IFN-based and IFN-free therapies in Japan, which are associated with differential risk of HCC after SVR. These differences can influence the incidence of HCC after SVR and should be taken into consideration when comparing IFN-based and IFN-free therapies in terms of hepatocarcinogenesis suppression with HCV eradication.

Evaluation of factors associated with relapse in telaprevir-based triple therapy for chronic hepatitis C.

BACKGROUND AND RATIONALE: Most patients with chronic hepatitis C show virological response to telaprevir-based triple therapy, and achieve an end-of-treatment response (ETR). However, some patients showing ETR develop virological relapse. This study was carried out to evaluate factors associated with relapse after triple therapy. MATERIALS AND METHODS: A prospective, multicentric study was conducted in chronic hepatitis C patients who received telaprevir-based triple therapy. We evaluated independent variables such as age, with or without cirrhosis, prior treatment response to interferon (IFN) therapy, IL28B genotype, core amino acid (aa) 70 mutation, drug adherence, white blood cell counts, hemoglobin level, and serum low-density lipoprotein (LDL) cholesterol level. The characteristics of the patients who relapsed after achieving ETR were compared with those who did not. RESULTS: Among 168 patients, 157 patients achieved ETR (93.5%) and 11 discontinued. Of these 157 patients, relapse occurred in 21 patients (13.4%). Nineteen patients (90.5%) of 21 relapsed patients had the IL28B non-TT genotype (P = 1.79 × 10 -9 ). Multivariate analysis identified core amino acid 70 [P = 0.018, crude odds ratio (OR): 6.927] and the IL28B genotype (P = 3.758 × 10 -5 , crude OR: 39.311) as significantly independent factors that influenced the relapse-related variables. Among the 49 patients with the IL28B non-TT, 18 patients had core aa70 mutation and 31 patients had core aa70 wild-type. In addition, 66.7% (12/18) of those with core aa70 mutation and 22.6% (7/31) of those with core aa70 wild-type developed relapse (P = 0.005). DISCUSSION: Core aa70 mutation and the IL28B non-TT genotype were identified as independent factors that influenced relapse after achievement of ETR for telaprevir-based triple therapy.

Changes in hepatitis C virus genotype distribution in Japan.

Genotypes are associated with the natural course of hepatitis C virus (HCV) infection and response to antiviral therapy for HCV. HCV genotype 1b has been the dominant genotype in Japan, where the prevention of HCV transmission through blood transfusion or nosocomial infection has been established since 1990. The distribution of HCV genotype was investigated based on patient's birth year in 5515 HCV-infected Japanese individuals at three institutions from different areas of Japan. At all three institutions, the proportion of HCV genotype 1b decreased and was <50% in individuals born after 1970. By contrast, the percentage of HCV genotype 2b increased in subsequent birth cohorts after 1920-1929. Significant changes in HCV genotype distribution were observed across Japan regardless of area.

Prospective study of kinesthesia after ACL reconstruction.

Previous studies used a variety of methods to assess kinesthesia, thus no consensus exists regarding kinesthetic adaptation after anterior cruciate ligament (ACL) reconstruction. This study prospectively examined whether kinesthesia is adapted after ACL reconstruction, and then discussed the actual angular velocity required to properly assess kinesthesia in ACL-reconstructed patients. 31 patients were evaluated using the threshold to detect passive motion (TTDPM) test, which was applied preoperatively, and at 3, 6, and 12 months following surgery. TTDPMs were measured at 15° or 45° of knee flexion toward both extension and flexion with angular velocities of 0.1°/s or 0.2°/s. ACL-reconstructed knees showed significantly impaired TTDPMs compared to healthy knees before the operation at 15° of knee flexion toward extension and at 45° of knee flexion toward both extension and flexion at 0.2°/s (15° of knee flexion toward extension, P=0.036; 45° of knee flexion toward extension, P=0.015; 45° of knee flexion toward flexion, P=0.030). However, there were no significant differences after 3 months of follow-up. On the basis of these results, applying 0.2°/s seems appropriate to assess TTDPM for patients with an ACL reconstruction, and kinesthesia is adapted within 12 months after the operation. Sensory function and biomechanical stability are also adapted following ACL reconstruction.

Nonalcoholic steatohepatitis in hepatocarcinoma: new insights about its prognostic role in patients treated with lenvatinib.

BACKGROUND: Hepatocellular carcinoma (HCC) treatment remains a big challenge in the field of oncology. The liver disease (viral or not viral) underlying HCC turned out to be crucial in determining the biologic behavior of the tumor, including its response to treatment. The aim of this analysis was to investigate the role of the etiology of the underlying liver disease in survival outcomes. PATIENTS AND METHODS: We conducted a multicenter retrospective study on a large cohort of patients treated with lenvatinib as first-line therapy for advanced HCC from both Eastern and Western institutions. Univariate and multivariate analyses were performed. RESULTS: Among the 1232 lenvatinib-treated HCC patients, 453 (36.8%) were hepatitis C virus positive, 268 hepatitis B virus positive (21.8%), 236 nonalcoholic steatohepatitis (NASH) correlate (19.2%) and 275 had other etiologies (22.3%). The median progression-free survival (mPFS) was 6.2 months [95% confidence interval (CI) 5.9-6.7 months] and the median overall survival (mOS) was 15.8 months (95% CI 14.9-17.2 months). In the univariate analysis for OS NASH-HCC was associated with longer mOS [22.2 versus 15.1 months; hazard ratio (HR) 0.69; 95% CI 0.56-0.85; P = 0.0006]. In the univariate analysis for PFS NASH-HCC was associated with longer mPFS (7.5 versus 6.5 months; HR 0.84; 95% CI 0.71-0.99; P = 0.0436). The multivariate analysis confirmed NASH-HCC (HR 0.64; 95% CI 0.48-0.86; P = 0.0028) as an independent prognostic factor for OS, along with albumin-bilirubin (ALBI) grade, extrahepatic spread, neutrophil-to-lymphocyte ratio, portal vein thrombosis, Eastern Cooperative Oncology Group (ECOG) performance status and alpha-fetoprotein. An interaction test was performed between sorafenib and lenvatinib cohorts and the results highlighted the positive predictive role of NASH in favor of the lenvatinib arm (P = 0.0047). CONCLUSION: NASH has been identified as an independent prognostic factor in a large cohort of patients with advanced HCC treated with lenvatinib, thereby suggesting the role of the etiology in the selection of patients for tyrosine kinase treatment. If validated, this result could provide new insights useful to improve the management of these patients.

Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin.

Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues.

We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat. To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied. cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli. Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end. The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies. Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity. They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability. Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition. The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis. There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum.

Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data.

As a new approach to predicting in vivo drug metabolism in humans, scaling of in vivo metabolic clearance from in vitro data obtained using human liver microsomes or hepatocytes is described in this review, based on the large number of literature data. Successful predictions were obtained for verapamil, loxtidine (lavoltidine), diazepam, lidocaine, phenacetin and some other compounds where CLint,in vitro is comparable with CLint,in vivo. On the other hand, for some metabolic reactions, differences in CLint,in vitro and CLint,in vivo greater than 5-fold were observed. The following factors are considered to be the cause of the differences: (1) metabolism in tissues other than liver, (2) incorrect assumption of rapid equilibrium of drugs between blood and hepatocytes, (3) presence of active transport through the sinusoidal membrane, and (4) interindividual variability. Furthermore, the possibility of predicting in vivo drug metabolic clearance from results obtained using a recombinant system of human P450 isozyme was described for a model compound, YM796, where the predicted metabolic clearances obtained from the recombinant system, taking account of the content of the P450 isozyme CYP3A4 in the human microsomes, were comparable with the observed clearances using human liver microsomes containing different amounts of CYP3A4. Even in the case where the first-pass metabolism exhibits nonlinearity, it appears to be possible to predict in vivo metabolic clearance from in vitro metabolic data.

Role of human cytochrome P450 3A4 in metabolism of medroxyprogesterone acetate.

Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced or recurrent breast cancer and endometrial cancer. The drug is extensively metabolized in the intestinal mucosa and in the liver. Cytochrome P450s (CYPs) involved in the metabolism of MPA were identified by using human liver microsomes and recombinant human CYPs. In this study, the overall metabolism of MPA was determined as the disappearance of the parent drug from an incubation mixture. The disappearance of MPA in human liver microsomes varied 2.6-fold among the 18 samples studied. The disappearance of MPA in the same panel of 18 human liver microsomes was significantly correlated with triazolam alpha-hydroxylase activity, a marker activity of CYP3A (r = 0.764; P < 0.001). Ketoconazole, an inhibitor of CYP3A4, potently inhibited the disappearance of MPA in 18 human liver microsomes. Anti-CYP3A antibody also inhibited 86% of the disappearance of MPA in human liver microsomes. Although sulfaphenazole (an inhibitor of CYP2C9) and S-mephenytoin (an inhibitor of CYP2C19) partially inhibited the disappearance of MPA, no effect of the anti-CYP2C antibody was observed. The disappearance of MPA did not correlate with either the activity metabolized via CYP2C9 (diclofenac 4'-hydroxylase activity) or the activity metabolized via CYP2C19 (S-mephenytoin 4'-hydroxylase activity). Among the 12 recombinant human CYPs (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) studied, only CYP3A4 showed metabolic activity of MPA. These results suggest that CYP3A4 is mainly involved in the overall metabolism of MPA in human liver microsomes.

Involvement of microsomal cytochrome P450 and cytosolic thymidine phosphorylase in 5-fluorouracil formation from tegafur in human liver.

Recently, we have reported that tegafur, an anticancer agent, is biotransformed into active drug 5-fluorouracil (5-FU) by cytochromes P450 1A2, 2A6, and 2C8 in human liver microsomes (T. Komatsu et al., Drug Metab. Dispos, 28: 1457-1463, 2000). Because the conversion of tegafur into 5-FU has also been reported to be catalyzed by cytosolic thymidine phosphorylase (dThdPase), the involvement of human liver microsomes and cytosol and individual differences in 5-FU formation from tegafur were investigated. In 14 human samples, the mean rates of 5-FU formation in liver microsomes were 5-fold and 2-fold higher than those in liver cytosol at substrate concentrations of 100 microM and 1 mM tegafur, respectively. In the presence of 5-chloro-2,4-dihydroxypyridine, a dihydropyrimidine dehydrogenase inhibitor, the rates of 5-FU formation by the combination of liver microsomes and cytosol showed 5- and 3-fold interindividual differences at 100 microM and 1 mM tegafur, respectively. Kinetic analysis of human liver cytosolic 5-FU formation indicated an apparent higher Km value (16 +/- 4 mM) than that of liver microsomes (1.8 +/- 0.3 mM) with similar Vmax values. Human liver cytosolic 5-FU formation was confirmed to be catalyzed by dThdPase with correlation and chemical inhibition studies. These results suggested that 5-FU formation from tegafur in human liver was mainly catalyzed by microsomal P450 at low concentrations of tegafur, but the contribution of cytosolic 5-FU formation by dThdPase would be important at high concentrations.

Effect of the antiplatelet drug dilazep dihydrochloride on urinary podocytes in patients in the early stage of diabetic nephropathy.

OBJECTIVE: To determine whether the antiplatelet drug dilazep dihydrochloride affects the number of urinary podocytes in diabetic patients with microalbuminuria. RESEARCH DESIGN AND METHODS: Fifty patients with type 2 diabetes and microalbuminuria (30 men and 20 women, mean age 48.6 years) and 30 age-matched control subjects (18 men and 12 women, mean age 49.2 years) were included in the study. No patients showed serum creatinine levels in excess of 2.0 mg/dl. Urinary podocytes were examined by immunofluorescence microscopy with monoclonal antibodies against podocalyxin. RESULTS: Urinary podocytes were detected in 18 of the 50 microalbuminuric diabetic patients (mean, 1.3 cells/ml). Urinary podocytes were not detected in the remaining 32 patients or in the 30 healthy control subjects. Diabetic patients positive for urinary podocytes were divided into 2 treatment groups: a dilazep dihydrochloride treatment group (300 mg/day; n = 9, group A) and a placebo group (n = 9, group B). Treatments were continued for 6 months. In group A, microalbuminuria decreased significantly from 146 +/- 42 to 86 +/- 28 microg/min (P < 0.01) and urinary podocytes also decreased from 1.3 +/- 0.8 to 0.4 +/- 0.2 cells/ml (P < 0.01). However, in group B, microalbuminuria and urinary podocytes changed little over the study period. CONCLUSIONS: Podocyte injury may occur in patients with early diabetic nephropathy, and dilazep dihydrochloride may be useful for preventing glomerular injury.

Relationship between weight change in young adulthood and the risk of NIDDM. The Sotetsu Study.

OBJECTIVE: To investigate the independent effect of weight change in young adulthood on the risk of prevalent NIDDM among middle-aged Japanese men. RESEARCH DESIGN AND METHODS: A case-control study was carried out in 895 male employees aged > or = 30 years of a railway company located in the vicinity of Tokyo. Adjusted odds ratios (ORs) were calculated for prevalent diabetes in each category of weight change (obtained from subjects' medical records) in young adulthood and adulthood. Adjustment for current age, initial BMI, and weight change in each age stratum was performed by the Mantel-Haenszel method or multiple logistic regression analysis. RESULTS: Weight change between 20 years of age and the age at maximum weight was not associated with the risk of NIDDM. Weight gain between 20 and 25 years of age was significantly and positively associated with the risk of NIDDM (OR 3.87 for gains > or = 10.0 kg, 2.53 for gains of 5.0-9.9%, and 3.73 for gains > or = 10.0%). On the other hand, moderate weight gain after 30 years of age was significantly inversely associated with NIDDM (OR 0.44 for gains of 5.0-9.9 kg, 0.15 for gains of 10.0-19.9%, and 0.38 for gains of 20.0-29.9%). CONCLUSIONS: Extreme weight gain between 20 and 25 years of age is a significant predictor of NIDDM, independent of current age, BMI at 20 years of age, and weight change within other age strata.

CYP2D6 is the principal cytochrome P450 responsible for metabolism of the histamine H1 antagonist promethazine in human liver microsomes.

To determine which cytochrome P450 form is involved in the promethazine [10-(2-dimethylaminopropyl) phenothiazine] metabolism, in vitro analysis using human liver microsomes were performed. Promethazine was mainly biotransformed to ring-hydroxylated, S-oxidized and N-demethylated metabolites. The promethazine hydroxylase in human liver microsomes was inhibited by SKF-525A, propranolol, sparteine, quinidine and anti-CYP2D6 serum suggesting involvement of a P450 related to CYP2D6. Lineweaver-Burk plots for the hydroxylation, S-oxidation and N-demethylation indicated that the hydroxylation occurred with a low K(m) value in human liver microsomes. Microsomes from genetically-engineered human B-lymphoblastoid cells expressing CYP2D6 hydroxylated promethazine most efficiently as compared to other P450 forms, indicating that it was the principal P450 responsible for the metabolism of promethazine in human liver microsomes. The inhibition of CYP2D6-catalysed bufuralol 1'-hydroxylase by various histamine H3 antagonists including promethazine suggested that promethazine and some other histamine H1 antagonists could be inhibitors of this P450 in human liver microsomes.

Ischemic flow threshold for extracellular glutamate increase in cat cortex.

Extracellular glutamate (Glu), cerebral blood flow (CBF), and auditory-evoked potentials (AEPs) were measured concurrently using microdialysis and hydrogen clearance in the auditory cortex of anesthetized cats during global ischemia of various severities. A threshold-type relationship was observed between extracellular Glu and CBF: Glu increased at CBF levels below about 20 ml/100 g/min. The Glu increase was related to the impairment of AEPs. The results suggest that Glu neurotoxicity is an important factor for ischemic neuronal injury even in penumbra.

GDP does not support activation of adenylate cyclase nor ADP-ribosylation of a guanine nucleotide binding protein by cholera toxin.

The actions of cholera toxin (i.e., activation of adenylate cyclase and ADP-ribosylation of a guanine nucleotide binding protein in purified membranes from rat liver) were GTP dependent. Neither of these actions of cholera toxin was reproduced with GDP. Simultaneous addition of ATP and MgCl2 along with GDP allowed cholera toxin to exert these actions. The role of GDP in adenylate cyclase regulation was discussed.

Estradiol up-regulates the stimulatory GTP-binding protein expression in the MCF-7 human mammary carcinoma cell line.

The effect of estradiol treatment of the human mammary carcinoma cell MCF-7 on the adenylyl cyclase system was examined. Treatment with 10 nM estradiol for 72 h increased the basal level of cAMP, and isoproterenol-, PGE2- or calcitonin-stimulated cAMP production. Estradiol also increased the response to cholera toxin but did not alter the response to forskolin. No significant change in growth rate was observed during the 72 h of estradiol treatment. In MCF-7 cell membranes the responsiveness to isoproterenol, PGE2, or cholera toxin was also enhanced by estradiol treatment. The cholera toxin-catalyzed ADP-ribosylation of Gs alpha in MCF-7 cell membranes was significantly increased by 72 h of treatment with estradiol. Consistent with this observation, the level of Gs alpha immunoreactivity was increased in the estradiol-treated cell membranes. On the other hand, pertussis toxin did not change the responsiveness to isoproterenol, PGE2 or calcitonin in either control or estradiol-treated cells. In addition, ADP-ribosylation with pertussis toxin also did not reveal any change in Gi. These results clearly indicate that Gs expression is under the control of estradiol, and that this effect may contribute to the increased sensitivity of hormone-stimulated adenylyl cyclase activities in MCF-7 cells.

Increased expression of nucleoside diphosphate kinases/nm23 in human diploid fibroblasts transformed by SV40 large T antigen or 60Co irradiation.

When the expression levels of nucleoside diphosphate (NDP) kinase/nm23 were examined in four human normal diploid fibroblast cell lines in comparison with their corresponding immortalized cells transformed by SV40 large T antigen or 60Co irradiation, mRNA levels of the two isoforms (NDP kinase A/nm23-H1, NDP kinase B/nm23-H2) were increased in the immortalized cell lines. The increase was found to be associated with increased translation products. Furthermore, the cell extracts prepared from these immortalized cell lines demonstrated slightly higher enzyme activity than those from their normal counterparts. Neither the growth state nor the in vitro aging largely affected their expression in a normal cell line (TIG-3) examined. The results suggest possible involvement of NDP kinases/nm23 in acquiring an infinite growth property of these cells.

Activation of nucleoside diphosphate kinase by mastoparan, a peptide isolated from wasp venom.

We have previously reported that GDP-bound alpha beta gamma-trimeric GTP-binding (G) proteins can be converted into the active GTP-bound form with nucleoside diphosphate (NDP) kinase and ATP, although its exact activation mechanism still remains to be resolved. In the present study, we investigated whether NDP kinase activity was modified by mastoparan, a wasp venom peptide that is known to activate G proteins as an agonist-receptor complex. The activity of NDP kinase measured by the formation of GTP from ATP and GDP was markedly stimulated, when the kinase was incubated with mastoparan. The concentration of mastoparan required for the activation was much lower than that observed for the peptide-induced activation of G proteins under similar assay conditions. There was also an increase in the phosphorylated intermediate of NDP kinase as well as the catalytic activity upon its incubation with mastoparan. These results suggest that mastoparan not only activates G proteins directly via guanine nucleotide exchange reaction but also stimulates NDP kinase activity.

Overexpression of nucleoside diphosphate kinases induces neurite outgrowth and their substitution to inactive forms leads to suppression of nerve growth factor- and dibutyryl cyclic AMP-induced effects in PC12D cells.

Whether nucleoside diphosphate kinase (NDPK) is involved in neuronal differentiation was investigated with special reference to its enzyme activity. Neurite outgrowth of PC12D cells induced by nerve growth factor or a cyclic AMP analog was suppressed to some extent when inactive NDPKs (the active site histidine 118 was replaced with alanine), not active forms, were transiently overexpressed. This suppression was more definite in their stably expressed clones. NDPKbeta-transfected clones and, to a lesser extent, NDPKalpha-transfected clones, but not inactive NDPK-transfected clones, extended neurites without differentiation inducers. These results imply that NDPKs may play a role by exerting their enzyme activity during differentiation of PC12 cells.

Comparison between radioreceptor assay and radioimmunoassay for the determination of dihydroergotoxine in rabbit plasma samples.

Two analytical methods, radioreceptor assay and radioimmunoassay, for the determination of dihydroergotoxine have been developed. Antiserum, providing sufficient sensitivity for the radioimmunoassay, was produced by immunizing rabbits with D-lysergic acid coupled to bovine serum albumin. Radioreceptor assay utilizing dopamine receptor was carried out to determine dihydroergotoxine and its pharmacologically active metabolites in rabbit plasma, and the result was compared with that obtained by radioimmunoassay. The values obtained in both assays were almost identical; it was, therefore, assumed that the plasma concentrations of dihydroergotoxine determined by the present radioimmunoassay reflects the amount of unchanged drug and its active metabolites.