Amyloid polymorphisms constitute distinct clouds of conformational variants in different etiological subtypes of Alzheimer's disease.

The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-ß peptide (Aß) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aß can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of ß-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aß nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aß plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aß-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aß among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aß conformation and clinical phenotype.

Aß seeding potency peaks in the early stages of cerebral ß-amyloidosis.

Little is known about the extent to which pathogenic factors drive the development of Alzheimer's disease (AD) at different stages of the long preclinical and clinical phases. Given that the aggregation of the ß-amyloid peptide (Aß) is an important factor in AD pathogenesis, we asked whether Aß seeds from brain extracts of mice at different stages of amyloid deposition differ in their biological activity. Specifically, we assessed the effect of age on Aß seeding activity in two mouse models of cerebral Aß amyloidosis (APPPS1 and APP23) with different ages of onset and rates of progression of Aß deposition. Brain extracts from these mice were serially diluted and inoculated into host mice. Strikingly, the seeding activity (seeding dose SD50) in extracts from donor mice of both models reached a plateau relatively early in the amyloidogenic process. When normalized to total brain Aß, the resulting specific seeding activity sharply peaked at the initial phase of Aß deposition, which in turn is characterized by a temporary several-fold increase in the Aß42/Aß40 ratio. At all stages, the specific seeding activity of the APPPS1 extract was higher compared to that of APP23 brain extract, consistent with a more important contribution of Aß42 than Aß40 to seed activity. Our findings indicate that the Aß seeding potency is greatest early in the pathogenic cascade and diminishes as Aß increasingly accumulates in brain. The present results provide experimental support for directing anti-Aß therapeutics to the earliest stage of the pathogenic cascade, preferably before the onset of amyloid deposition.

Linking above- and belowground traits to soil and climate variables: an integrated database on China's grassland species.

Knowledge of plant functional traits and trait-environment interactions is important for characterizing species strategies and understanding ecological processes. However, comprehensive field data on both above- and belowground traits, together with their environmental variables are scarce. Biome-scale studies are particularly lacking. Here we present two large-scale data sets that include functional traits of leaves and fine roots and their corresponding soil and climatic variables in China's grasslands. Leaf, fine root, and soil samples were collected in three biogeographic regions: temperate grassland on the Inner Mongolia Plateau, alpine grassland on the Tibetan Plateau, and mountain grassland in the Xinjiang mountain areas. Field data were collected over two periods. The first data set collected between 2003 and 2004 includes 13 foliar traits (leaf mass per area, LMA; photosynthetic nitrogen use efficiency, PNUE; water use efficiency, WUE; stomatal conductance for water vapor, Gs; transpiration rate, TR; mass- and area-based photosynthetic capacity, Amass and Aarea; mass- and area-based carbon concentrations, Cmass and Carea; nitrogen concentrations, Nmass and Narea; and phosphorus concentrations, Pmass and Parea) for 170 species at 173 sites. The second data set collected between 2006 and 2007 includes six sets of analogous traits for both leaves and fine roots (C, N, and P concentrations; leaf thickness/root diameter; specific leaf area, SLA; specific root length, SRL; and tissue density) for 139 species at 82 sites, along with soil attributes (soil total and organic carbon, STC and SOC; total and available N, STN and SAN; total and available P, STP and SAP; pH, bulk density, and moisture). Moreover, associated information was also gathered, including geographical location (latitude, longitude, and altitude), climate (mean annual temperature, MAT; mean annual precipitation, MAP; growing season temperature, GST; growing season precipitation, GSP; potential evapotranspiration, PET; and actual evapotranspiration, AET) and site descriptions (vegetation and soil types). The data sets are unique because they integrate plant above- and belowground traits, climate, and soil factors over broad regional, elevational, and taxonomic ranges in understudied regions (e.g., the Tibetan Plateau). This is the only database on China's grassland species for unrestricted global access. These data sets will make a valuable contribution to future large-scale trait-based ecological studies.

Membrane pathology and microglial activation of mice expressing membrane anchored or membrane released forms of Aß and mutated human Alzheimer's precursor protein (APP).

AIMS: Alzheimer's disease and the transmissible spongiform encephalopathies or prion diseases accumulate misfolded and aggregated forms of neuronal cell membrane proteins. Distinctive membrane lesions caused by the accumulation of disease-associated prion protein (PrP(d)) are found in prion disease but morphological changes of membranes are not associated with Aß in Alzheimer's disease. Membrane changes occur in all prion diseases where PrP(d) is attached to cell membranes by a glycosyl-phosphoinositol (GPI) anchor but are absent from transgenic mice expressing anchorless PrP(d). Here we investigate whether GPI membrane attached Aß may also cause prion-like membrane lesions. METHODS: We used immunogold electron microscopy to determine the localization and pathology of Aß accumulation in groups of transgenic mice expressing anchored or unanchored forms of Aß or mutated human Alzheimer's precursor protein. RESULTS: GPI attached Aß did not replicate the membrane lesions of PrP(d). However, as with PrP(d) in prion disease, Aß peptides derived from each transgenic mouse line initially accumulated on morphologically normal neurite membranes, elicited rapid glial recognition and neurite Aß was transferred to attenuated microglial and astrocytic processes. CONCLUSIONS: GPI attachment of misfolded membrane proteins is insufficient to cause prion-like membrane lesions. Prion disease and murine Aß amyloidosis both accumulate misfolded monomeric or oligomeric membrane proteins that are recognized by glial processes and acquire such misfolded proteins prior to their accumulation in the extracellular space. In contrast to prion disease where glial cells efficiently endocytose PrP(d) to endolysosomes, activated microglial cells in murine Aß amyloidosis are not as efficient phagocytes.

Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells.

Under most physiological circumstances, monocytes are excluded from parenchymal CNS tissues. When widespread monocyte entry occurs, their numbers decrease shortly after engraftment in the presence of microglia. However, some disease processes lead to focal and selective loss, or dysfunction, of microglia, and microglial senescence typifies the aged brain. In this regard, the long-term engraftment of monocytes in the microglia-depleted brain remains unknown. Here, we report a model in which a niche for myeloid cells was created through microglia depletion. We show that microglia-depleted brain regions of CD11b-HSVTK transgenic mice are repopulated with new Iba-1-positive cells within 2 wk. The engrafted cells expressed high levels of CD45 and CCR2 and appeared in a wave-like pattern frequently associated with blood vessels, suggesting the engrafted cells were peripheral monocytes. Although two times more numerous and morphologically distinct from resident microglia up to 27 wk after initial engraftment, the overall distribution of the engrafted cells was remarkably similar to that of microglia. Two-photon in vivo imaging revealed that the engrafted myeloid cells extended their processes toward an ATP source and displayed intracellular calcium transients. Moreover, the engrafted cells migrated toward areas of kainic acid-induced neuronal death. These data provide evidence that circulating monocytes have the potential to occupy the adult CNS myeloid niche normally inhabited by microglia and identify a strong homeostatic drive to maintain the myeloid component in the mature brain.

Membrane-anchored Aß accelerates amyloid formation and exacerbates amyloid-associated toxicity in mice.

Pathological, genetic, and biochemical hallmarks of Alzheimer's disease (AD) are linked to amyloid-ß (Aß) peptide aggregation. Especially misfolded Aß42 peptide is sufficient to promote amyloid plaque formation. However, the cellular compartment facilitating the conversion of monomeric Aß to aggregated toxic Aß species remains unknown. In vitro models suggest lipid membranes to be the driving force of Aß conversion. To this end, we generated two novel mouse models, expressing either membrane-anchored or nonanchored versions of the human Aß42 peptide. Strikingly, membrane-anchored Aß42 robustly accelerated Aß deposition and exacerbated amyloid-associated toxicity upon crossing with Aß precursor protein transgenic mice. These in vivo findings support the hypothesis that Aß-membrane interactions play a pivotal role in early-onset AD as well as neuronal damage and provide evidence to study Aß-membrane interactions as therapeutic targets.

Aß seeds resist inactivation by formaldehyde.

Cerebral ß-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated ß-amyloid (Aß) into young, pre-depositing Aß precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated Aß itself. Here we report that the ß-amyloid-inducing activity of Alzheimer's disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated Aß are maintained in fixed tissues. The resistance of Aß seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of Aß aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

Characterization of Drug Release from Mesoporous SiO2-Based Membranes with Variable Pore Structure and Geometry.

Transdermal drug delivery systems (TDDSs) play important roles in therapy due to distinct advantages over other forms and types of drug application. While common TDDS patches mainly consist of polymeric matrices so far, inorganic carriers show numerous advantages such as high mechanical stability, possible re-use and re-loading of drugs, and a broad chemical compatibility with therapeutically relevant compounds and chemical enhancers. Mesoporous glasses can be prepared in different monolithic shapes, and offer a particularly wide range of possible pore volumes, pore diameters, and specific surface areas. Further, they show high loading capacities and favorable physical, technical, and biological properties. Here, we explored for the first time monolithic SiO2-based carriers as sustained release systems of therapeutic drugs. In an ideally stirred vessel as model system, we systematically analyzed the influence of pore diameter, pore volume, and the dimensions of glass monoliths on the loading and sustained release of different drugs, including anastrozole, xylazine, imiquimod, levetiracetam, and flunixin. Through multilinear regression, we calculated the influence of different parameters on drug loading and diffusion coefficients. The systematic variation of the mesoporous glass properties revealed pore volumes and drug loading concentrations, but not pore diameter or pore surface area as important parameters of drug loading and release kinetics. Other relevant effectors include the occurrence of lateral diffusion within the carrier and drug-specific properties such as adsorption. The structure-property relationships derived from our data will allow further fine-tuning of the systems according to their desired properties as TDDS, thus guiding towards optimal systems for their use in transdermal drug applications.

Experimental evidence for temporal uncoupling of brain Aß deposition and neurodegenerative sequelae.

Brain Aß deposition is a key early event in the pathogenesis of Alzheimer´s disease (AD), but the long presymptomatic phase and poor correlation between Aß deposition and clinical symptoms remain puzzling. To elucidate the dependency of downstream pathologies on Aß, we analyzed the trajectories of cerebral Aß accumulation, Aß seeding activity, and neurofilament light chain (NfL) in the CSF (a biomarker of neurodegeneration) in Aß-precursor protein transgenic mice. We find that Aß deposition increases linearly until it reaches an apparent plateau at a late age, while Aß seeding activity increases more rapidly and reaches a plateau earlier, coinciding with the onset of a robust increase of CSF NfL. Short-term inhibition of Aß generation in amyloid-laden mice reduced Aß deposition and associated glial changes, but failed to reduce Aß seeding activity, and CSF NfL continued to increase although at a slower pace. When short-term or long-term inhibition of Aß generation was started at pre-amyloid stages, CSF NfL did not increase despite some Aß deposition, microglial activation, and robust brain Aß seeding activity. A dissociation of Aß load and CSF NfL trajectories was also found in familial AD, consistent with the view that Aß aggregation is not kinetically coupled to neurotoxicity. Rather, neurodegeneration starts when Aß seeding activity is saturated and before Aß deposition reaches critical (half-maximal) levels, a phenomenon reminiscent of the two pathogenic phases in prion disease.

Acute targeting of pre-amyloid seeds in transgenic mice reduces Alzheimer-like pathology later in life.

Amyloid-ß (Aß) deposits are a relatively late consequence of Aß aggregation in Alzheimer's disease. When pathogenic Aß seeds begin to form, propagate and spread is not known, nor are they biochemically defined. We tested various antibodies for their ability to neutralize Aß seeds before Aß deposition becomes detectable in Aß precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aß assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aß deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aß seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer's disease-currently defined as Aß deposition without clinical symptoms-may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.

Identification and quantitation of the N-acetyl-L-cysteine S-conjugates of bendamustine and its sulfoxides in human bile after administration of bendamustine hydrochloride.

We recently reported the detection of mercapturic acid pathway metabolites of bendamustine, namely, cysteine S-conjugates in human bile, which are supposed to subsequently undergo further metabolism. In this study, we describe the identification and quantitation of consecutive bendamustine metabolites occurring in human bile using authentic reference standards and the synthesis and structural confirmation of these compounds. Mass spectrometry data along with high-performance liquid chromatography retention data (fluorescence detection) of the synthetic reference standards were consistent with those of the metabolites found in human bile after administration of bendamustine hydrochloride to cancer patients. Analysis of the purified synthetic reference compounds showed a purity of at least 95%. Structural confirmation was achieved by one- and two-dimensional proton as well as carbon-13 NMR spectroscopy and mass spectrometry. A total of 16 bendamustine-related compounds were detected in the bile of patients, 11 of them were recovered as conjugates. Eight conjugates have been structurally confirmed as novel mercapturic acids and sulfoxides. Biliary excretion of the sulfoxides was twice that of the mercapturate precursors. Glutathione S-conjugates of bendamustine have not been detected in bile samples, indicating rapid enzymatic cleavage in humans. Both the lack of glutathione (GSH) conjugates and occurrence of diastereomeric sulfoxides emphasize species-related differences in the GSH conjugation of bendamustine between humans and rats. The total amount recovered in the bile as the sum of all conjugates over the period of 24 h after dosing averaged 5.2% of the administered dose. The question of whether the novel metabolites contribute to urinary excretion should be a target of future investigations.

Pharmacokinetic evaluation of a transdermal anastrozole-in-adhesive formulation.

BACKGROUND AND OBJECTIVE: Anastrozole is a well-established active pharmaceutical ingredient (API) used for the treatment of hormone-sensitive breast cancer (BC) in postmenopausal women. However, treatment with the only available oral formulation is often associated with concentration-dependent serious side effects such as hot flashes, fatigue, muscle and joint pain, nausea, diarrhea, headache, and others. In contrast, a sustained-release system for the local application of anastrozole should minimize these serious adverse drug reactions. METHODS: Anastrozole-in-adhesive transdermal drug delivery systems (TDDS) were developed offering efficient loading, avoidance of inhomogeneity or crystallization of the drug, the desired controlled release kinetics, storage stability, easy handling, mechanical stability, and sufficient stickiness on the skin. In vitro continuous anastrozole release profiles were studied in Franz diffusion cells. In vivo, consecutive drug plasma kinetics from the final anastrozole transdermal system was tested in beagle dogs. For drug analysis, a specific validated liquid chromatography- mass spectrometry method using fragment ion detection was developed and validated. RESULTS: After efficient drug loading, a linear and sustained 65% drug release from the TDDS over 48 h was obtained. In vivo data showed a favorable anastrozole plasma concentration-time course, avoiding side effect-associated peak concentrations as obtained after oral administration but matching therapeutic plasma levels up to 72 h. CONCLUSION: These results provide the basis for establishing the transdermal application of anastrozole with improved pharmacokinetics and drug safety as novel therapeutic approach and promising option to treat human BC by decreasing the high burden of unwanted side effects.

Characterization of two phase I metabolites of bendamustine in human liver microsomes and in cancer patients treated with bendamustine hydrochloride.

PURPOSE: The metabolism of bendamustine (BM) hydrochloride, a bifunctional alkylator containing a heterocyclic ring, was investigated in vitro and in vivo for identification of cytochromes P450 (CYP) involved in the formation of two phase I metabolites, structural confirmation of these previously unidentified metabolites and assessment of their cytotoxic effect in relation to the parent compound. METHODS: Potential metabolites of BM were synthesized and structurally characterized by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. In vitro metabolism of BM hydrochloride in human hepatic microsomes was conducted to identify the CYP450 isoenzymes involved in the oxidative metabolism of BM. Samples from cancer patients after treatment with BM hydrochloride and microsomal preparations were analyzed by LC-MS and HPLC with fluorescence detection. The cytotoxic effect of the metabolites was analyzed in several lymphoma cell lines and peripheral blood lymphocytes and compared with that of the parent compound using an MTT assay. RESULTS: LC-MS as well as HPLC with fluorescence detection revealed hydroxylation of the methylene carbon at the C-4 position of the butanoic acid side chain and N-demethylation of the benzimidazole skeleton as the main metabolic pathways in human liver microsomes. Isoform-specific chemical inhibitors and correlation analysis pointed to CYP1A2 as the prominent enzyme in BM oxidation. The rate of formation for both metabolites correlated (r=0.931 and 0.933) with the activity of CYP1A2 and there were no other notable correlations with any of the other CYPs. In addition, both metabolites were identified in plasma, urine, and bile samples from cancer patients under BM hydrochloride therapy as shown by comparison with chromatograms obtained from the authentic reference standards. Cytotoxic activity observed for gamma-hydroxy BM was approximately equivalent to that obtained for the parental compound BM. N-demethyl BM displays five to tenfold less cytotoxic activity than BM. CONCLUSION: The results indicate that CYP1A2-catalyzed N-dealkylation and gamma hydroxylation are the major routes for BM phase I metabolism producing two metabolites less or similarly toxic than the parent compound. In contrast to the metabolic pathways of the structurally related chlorambucil, no beta-oxidation of the butanoic acid side chain leading to enhanced toxicity was detected for BM.

Increasing temperature reduces the coupling between available nitrogen and phosphorus in soils of Chinese grasslands.

Changes in climatic conditions along geographical gradients greatly affect soil nutrient cycling processes. Yet how climate regimes such as changes in temperature influence soil nitrogen (N) and phosphorus (P) concentrations and their stoichiometry is not well understood. This study investigated the spatial pattern and variability of soil N and P availability as well as their coupling relationships at two soil layers (0-10 and 10-20 cm) along a 4000-km climate transect in two grassland biomes of China, the Inner Mongolian temperate grasslands and the Tibetan alpine grasslands. Our results found that in both grasslands, from cold to warm sites the amounts of soil total N, total P and available P all decreased. By contrast, the amount of available N was positively related to mean annual temperature in the Tibetan grasslands. Meanwhile, with increasing temperature ratio of available N to P significantly increased but the linear relationship between them was considerably reduced. Thus, increasing temperature may not only induce a stoichiometric shift but also loose the coupling between available N and P. This N-P decoupling under warmer conditions was more evident in the Tibetan alpine grasslands where P limitation might become more widespread relative to N as temperatures continue to rise.

Age-Dependent Protein Aggregation Initiates Amyloid-ß Aggregation.

Aging is the most important risk factor for neurodegenerative diseases associated with pathological protein aggregation such as Alzheimer's disease. Although aging is an important player, it remains unknown which molecular changes are relevant for disease initiation. Recently, it has become apparent that widespread protein aggregation is a common feature of aging. Indeed, several studies demonstrate that 100s of proteins become highly insoluble with age, in the absence of obvious disease processes. Yet it remains unclear how these misfolded proteins aggregating with age affect neurodegenerative diseases. Importantly, several of these aggregation-prone proteins are found as minor components in disease-associated hallmark aggregates such as amyloid-ß plaques or neurofibrillary tangles. This co-localization raises the possibility that age-dependent protein aggregation directly contributes to pathological aggregation. Here, we show for the first time that highly insoluble proteins from aged Caenorhabditis elegans or aged mouse brains, but not from young individuals, can initiate amyloid-ß aggregation in vitro. We tested the seeding potential at four different ages across the adult lifespan of C. elegans. Significantly, protein aggregates formed during the early stages of aging did not act as seeds for amyloid-ß aggregation. Instead, we found that changes in protein aggregation occurring during middle-age initiated amyloid-ß aggregation. Mass spectrometry analysis revealed several late-aggregating proteins that were previously identified as minor components of amyloid-ß plaques and neurofibrillary tangles such as 14-3-3, Ubiquitin-like modifier-activating enzyme 1 and Lamin A/C, highlighting these as strong candidates for cross-seeding. Overall, we demonstrate that widespread protein misfolding and aggregation with age could be critical for the initiation of pathogenesis, and thus should be targeted by therapeutic strategies to alleviate neurodegenerative diseases.

Highly potent intracellular membrane-associated Aß seeds.

An early event in Alzheimer's disease (AD) pathogenesis is the formation of extracellular aggregates of amyloid-ß peptide (Aß), thought to be initiated by a prion-like seeding mechanism. However, the molecular nature and location of the Aß seeds remain rather elusive. Active Aß seeds are found in crude homogenates of amyloid-laden brains and in the soluble fraction thereof. To analyze the seeding activity of the pellet fraction, we have either separated or directly immunoisolated membranes from such homogenates. Here, we found considerable Aß seeding activity associated with membranes in the absence of detectable amyloid fibrils. We also found that Aß seeds on mitochondrial or associated membranes efficiently induced Aß aggregation in vitro and seed ß-amyloidosis in vivo. Aß seeds at intracellular membranes may contribute to the spreading of Aß aggregation along neuronal pathways and to the induction of intracellular pathologies downstream of Aß.

Persistence of Aß seeds in APP null mouse brain.

Cerebral ß-amyloidosis is induced by inoculation of Aß seeds into APP transgenic mice, but not into App(-/-) (APP null) mice. We found that brain extracts from APP null mice that had been inoculated with Aß seeds up to 6 months previously still induced ß-amyloidosis in APP transgenic hosts following secondary transmission. Thus, Aß seeds can persist in the brain for months, and they regain propagative and pathogenic activity in the presence of host Aß.

Measurement of 4-hydroxylation of ifosfamide in human liver microsomes using the estimation of free and protein-bound acrolein and codetermination of keto- and carboxyifosfamide.

PURPOSE: The aim of the present study was to determine the turnover (4-hydroxylation and N-dechloroethylation) of ifosfamide in a total of 25 human liver microsomal preparations in which the codetermination of keto- and carboxyifosfamide as well as the calculation of free and protein-bound acrolein was carried out for the first time. METHODS: The 4-hydroxylation of ifosfamide was estimated by using acrolein (free and protein-bound) and a newly developed procedure involving the codetermination of keto- and carboxyifosfamide (LC/MS). The ifosfamide N-dechloroethylation was determined as the sum of 2- and 3-dechloroethylifosfamide (LC/MS). RESULTS: Using the usual estimation of liberated free acrolein in 25 human liver microsomal preparations, the 4-hydroxylation of ifosfamide amounted to 0.28+/-0.16 nmol/min. nmol(P450). However, after calculating the 4-hydroxylation as the sum of free and protein-bound acrolein and keto- and carboxyifosfamide, a ninefold higher activity (2.40+/-0.73 nmol/min. nmol(P450)) was found. The percentage of the inactive metabolites keto- (25/25) and carboxyifosfamide (5/25) in the 4-hydroxylation amounted to only 0.79-5.25% (mean 2.90%). The ifosfamide N-dechloroethylation (mean 0.21+/-0.11 nmol/min. nmol(P450)) determined as the sum of 2- and 3-dechloroethylifosfamide was estimated as 8.3+/-4.3% of the total ifosfamide turnover. The application of the relative substrate-activity factor (RSF)-approach and the calculation of the contribution of various isoforms in the ifosfamide 4-hydroxylation yielded the following results: CYP 3A4: 58+/-31%, CYP 2A6: 25+/-15%, and CYP 2C9: 5+/-2% of the total measured 4-hydroxylation. A correlation between 4-hydroxylation and the N-dechloroethylation rates of ifosfamide and the activities of isoenzymes indicates the involvement of both CYP 3A4 ( P=0.026) and CYP 2C9 ( P=0.012) in the 4-hydroxylation reaction and of CYP 3A4 ( P<0.01) in the N-dechloroethylation reaction. CONCLUSIONS: The estimation of protein-bound acrolein should be included in the calculation of the ifosfamide 4-hydroxylation besides liberated free acrolein. Because of the small amounts of the inactive metabolites keto- and carboxyifosfamide, the exclusive determination of acrolein only (free and protein-bound) seems to suffice for the calculation of total ifosfamide hydroxylation. Using this method the hepatic in vitro turnover of ifosfamide was estimated as 92% for 4-hydroxylation (CYP 3A4 and CYP 2A6 mediated) and 8% for N-dechloroethylation (CYP 3A4 mediated), and in this way, a relative overestimation of the N-dechloroethylation of ifosfamide on the whole metabolism is avoided.

Investigations on the pharmacokinetics of trofosfamide and its metabolites-first report of 4-hydroxy-trofosfamide kinetics in humans.

Trofosfamide (TRO), like cyclophosphamide (CYCLO) and ifosfamide (IFO), is a prodrug oxazaphosphorine derivative that requires hepatic biotransformation to form the cytotoxically active 4-hydroxy derivative (4-hydroxy-TRO). Individual 4-hydroxyoxazaphosphorines and 4-hydroxy-TRO itself have not been demonstrated in humans up to now. For investigation of the principal pharmacokinetics of TRO and its metabolites, six tumour patients (49-65 years of age, Karnofsky index >70%) with normal liver and renal function were given a single oral dose of 600 mg/m(2) TRO. Plasma was sampled using a bedside technique. Individual 4-hydroxyoxazaphosphorines and TRO together with further metabolites were determined by a specially developed HPLC-UV method and a HPLC-MS method, respectively. With a short apparent half-life (1.2 h) and high apparent clearance (Cl/F 4.0 l/min), TRO was very quickly eliminated from plasma and highly converted to its metabolites, mainly 4-hydroxy-TRO and IFO. In relation to the AUC values of TRO (1.0) the following molar quotients were calculated: 1.59 (4-hydroxy-TRO), 0.40 (4-hydroxy-IFO), 6.90 (IFO) and 0.74 (CYCLO). C(max) values were in the range 10-13 micromol/l for TRO, 4-hydroxy-TRO and IFO and in the range 1.5-4.0 micromol/l for CYCLO, 2- and 3-dechloroethyl-IFO and 4-hydroxy-IFO. Kinetic data indicate that 4-hydroxy-IFO is formed by both hydroxylation of TRO and exocyclic N-dechloroethylation of 4-hydroxy-TRO. 4-hydroxy-CYCLO was not detected above the quantification limit of the method. Only mild haemodepressive side effects were observed after oral administration of 600 mg/m(2) TRO. In relation to known data for IFO, TRO is much more 4-hydroxylated than IFO. The high 4-hydroxy-TRO/TRO ratio found suggests that TRO is a promising tumourstatic agent.