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The active enantiomer R-Praziquantel (PZQ) shows a clinically lower relative exposure when administered enantiomerically pure compared with a racemic form. We investigated the hypothesis that enantiomer-enantiomer interactions on cytochrome P450 (P450) enzymes could explain this observation and aimed to further deepen the understanding of PZQ metabolism. First, in an in vitro metabolite profiling study, the formation of multiple metabolites per P450, together with an observed interconversion of cis-4'-OH-PZQ to trans-4'-OH-PZQ in human hepatocytes, pointed out the inadequacy of measuring metabolite formation in kinetic studies. Thus, a substrate depletion approach to study PZQ enantiomeric interactions was applied. Second, an abundant CYP3A4 metabolite found in previous studies was structurally characterized. Third, substrate depletion methodologies were applied to determine P450 enzyme kinetics of PZQ and to further estimate enantiomer-enantiomer inhibitory parameters. A competitive inhibition between PZQ enantiomers for CYP2C9, 2C19, 3A4, and 3A5 was revealed. Analyses considering the clearance of only one or both enantiomers provided comparable enantiomer-enantiomer inhibition estimates. To conclude, this paper provides new insights into PZQ metabolic profile to enable a better understanding of enantioselective pharmacokinetics using substrate depletion-based methods. SIGNIFICANCE STATEMENT: In this study, enantiomer-enantiomer interactions of praziquantel on cytochrome P450 metabolizing enzymes are investigated via substrate depletion measurement using modeling methods. Together with new insights into the praziquantel metabolism, this work provides a novel data set to understand its pharmacokinetics.

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry.

Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [(3)H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [(3)H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [(3)H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [(3)H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [(3)H]-SSB siRNA.

We present precise iron stable isotope ratios measured by multicollector-ICP mass spectrometry (MC-ICP-MS) of human red blood cells (erythrocytes) and blood plasma from 12 healthy male adults taken during a clinical study. The accurate determination of stable isotope ratios in plasma first required substantial method development work, as minor iron amounts in plasma had to be separated from a large organic matrix prior to mass-spectrometric analysis to avoid spectroscopic interferences and shifts in the mass spectrometer's mass-bias. The (56)Fe/(54)Fe ratio in erythrocytes, expressed as permil difference from the "IRMM-014" iron reference standard (δ(56/54)Fe), ranges from -3.1‰ to -2.2‰, a range typical for male Caucasian adults. The individual subject erythrocyte iron isotope composition can be regarded as uniform over the 21 days investigated, as variations (±0.059 to ±0.15‰) are mostly within the analytical precision of reference materials. In plasma, δ(56/54)Fe values measured in two different laboratories range from -3.0‰ to -2.0‰, and are on average 0.24‰ higher than those in erythrocytes. However, this difference is barely resolvable within one standard deviation of the differences (0.22‰). Taking into account the possible contamination due to hemolysis (iron concentrations are only 0.4 to 2 ppm in plasma compared to approx. 480 ppm in erythrocytes), we model the pure plasma δ(56/54)Fe to be on average 0.4‰ higher than that in erythrocytes. Hence, the plasma iron isotope signature lies between that of the liver and that of erythrocytes. This difference can be explained by redox processes involved during cycling of iron between transferrin and ferritin.

Absorption, distribution, metabolism, and excretion properties of two unformulated model short interfering RNA (siRNAs) were determined using a single internal [(3)H]-radiolabeling procedure, in which the full-length oligonucleotides were radiolabeled by Br/(3)H -exchange. Tissue distribution, excretion, and mass balance of radioactivity were investigated in male CD-1 mice after a single intravenous administration of the [(3)H]siRNAs, at a target dose level of 5 mg/kg. Quantitative whole-body autoradiography and liquid scintillation counting techniques were used to determine tissue distribution. Radiochromatogram profiles were determined in plasma, tissue extracts, and urine. Metabolites were separated by liquid chromatography and identified by radiodetection and high-resolution accurate mass spectrometry. In general, there was little difference in the distribution of total radiolabeled components after administration of the two unformulated [(3)H]siRNAs. The radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated at later time points (24 and 48 hours for [(3)H]MRP4 (multidrug resistance protein isoform 4) and [(3)H]SSB (Sjögren Syndrome antigen B) siRNA, respectively). After an initial rapid decrease, concentrations of total radiolabeled components in dried blood decreased at a much slower rate. A nearly complete mass balance was obtained for the [(3)H]SSB siRNA, and renal excretion was the main route of elimination (38%). The metabolism of the two model siRNAs was rapid and extensive. Five minutes after administration, no parent compound could be detected in plasma. Instead, radiolabeled nucleosides resulting from nuclease hydrolysis were observed. In the metabolism profiles obtained from various tissues, only radiolabeled nucleosides were found, suggesting that siRNAs are rapidly metabolized and that the distribution pattern of total radiolabeled components can be ascribed to small molecular weight metabolites.

The chemical modification 2′‐O‐methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)‐properties of tritium‐labeled 2′‐O‐methyluridine, following a single intravenous dose to male CD‐1 mice. The single intravenous administration of [5‐3H]‐2′‐O‐methyluridine was well tolerated in mice. Radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated throughout the observation period of 48 h. After an initial rapid decline, blood concentrations of total radiolabeled components declined at a much slower rate. [3H]‐2′‐O‐Methyluridine represented a minor component of the radioactivity in plasma (5.89% of [3H]‐AUC 0‐48 h). Three [3H]‐2′‐O‐methyluridine metabolites namely uridine (M1), cytidine (M2), and uracil (M3) were the major circulating components representing 32.8%, 8.11%, and 23.6% of radioactivity area under the curve, respectively. The highest concentrations of total radiolabeled components and exposures were observed in kidney, spleen, pineal body, and lymph nodes. The mass balance, which is the sum of external recovery of radioactivity in excreta and remaining radioactivity in carcass and cage wash, was complete. Renal excretion accounted for about 52.7% of the dose with direct renal excretion of the parent in combination with metabolism to the endogenous compounds cytidine, uracil, cytosine, and cytidine.

Modern accelerator mass spectrometry (AMS) methods enable the routine application of this technology in drug development. By the administration of a (14)C-labelled microdose or microtrace, pharmacokinetic (PK) data, such as mass balance, metabolite profiling, and absolute bioavailability (AB) data, can be generated easier, faster, and at lower costs. Here, we emphasize the advances and impact of this technology for pharmaceutical companies. The availability of accurate intravenous (iv) PK and human absorption, distribution, metabolism, and excretion (ADME) information, even before or during Phase I trials, can improve the clinical development plan. Moreover, applying the microtrace approach during early clinical development might impact the number of clinical pharmacology and preclinical safety pharmacology studies required, and shorten the overall drug discovery program.

A phase 1, open-label, non-randomized, single center study was conducted to determine the pharmacokinetics, distribution, metabolism, elimination, and mass balance of patupilone in patients with advanced solid tumors. Five patients with advanced solid tumors received 10 mg/m(2) (1.1 MBq) of (14) C-radiolabeled patupilone at cycle 1 as a 20-minute intravenous infusion every 3 weeks until disease progression. Sequential samples of blood/plasma were taken for 3 weeks and urine and fecal samples were collected for seven days after the first dose of patupilone. Patupilone blood levels decreased rapidly after the infusion. The compound showed a large volume of distribution (Vss: 2242 L). The main radiolabeled component in blood was patupilone itself, accompanied by the lactone hydrolysis products that are unlikely to contribute to the pharmacological effect of patupilone. The blood clearance of patupilone was relatively low at 14 L/h. The administered radioactivity dose was excreted slowly (46 % of dose up to 168 h) but ultimately accounted for 91 % of the dose by extrapolation. The fecal excretion of radioactivity was 2-3 times higher than the urinary excretion consistent with hepato-biliary elimination. Three patients had progressive disease and two patients had stable disease as their best response. Patupilone was generally well tolerated in patients with advanced solid tumors with no newly occurring safety events compared to previous clinical studies. In adult solid tumor patients, intravenous radiolabeled patupilone undergoes extensive metabolism with fecal excretion of radioactive metabolites predominating over renal excretion.

To determine the pharmacokinetics of the p110α-selective inhibitor alpelisib (BYL719) in humans, to identify metabolites in plasma and excreta, and to characterize pathways of biotransformation. Four healthy male volunteers received a single oral dose of [14C]-labeled alpelisib (400 mg, 2.78 MBq). Blood, urine, and feces samples were collected throughout the study. Total radioactivity was measured by liquid scintillation counting, and metabolites were quantified and identified by radiometry and mass spectrometry. Complementary in vitro experiments characterized the hydrolytic, oxidative, and conjugative enzymes involved in metabolite formation. Over 50 % of [14C] alpelisib was absorbed, with a T max of 2 h and an elimination half-life from plasma of 13.7 h. Over the first 12 h, exposure to alpelisib and the primary metabolite M4 was 67.9 and 26.7 % of total drug-related material in circulation, respectively. Mass balance was achieved, with 94.5 % of administered radioactivity recovered in excreta. In total, 38.2 % of alpelisib was excreted unchanged, while 39.5 % was excreted as M4. Based on the excreta pools analyzed, excretion occurred mainly via feces (79.8 % of administered dose); 13.1 % was excreted via urine. In vitro experiments showed that spontaneous and enzymatic hydrolysis contributed to M4 formation, while CYP3A4-mediated oxidation and UGT1A9-mediated glucuronidation formed minor metabolites. Alpelisib was well tolerated, and no new safety concerns were raised during this study. Alpelisib was rapidly absorbed and cleared by multiple metabolic pathways; the primary metabolite M4 is pharmacologically inactive. Alpelisib has limited potential for drug–drug interactions and is therefore a promising candidate for combination therapy.

Solifenacin succinate (YM905) is a new, once-daily, orally administered muscarinic receptor antagonist designed to treat overactive bladder. The metabolism of solifenacin involves hepatic cytochrome P450 (CYP) 3A4; therefore, the pharmacokinetics of solifenacin may be affected by drugs that inhibit CYP3A4. This study aimed to examine the effects of co-administration of ketoconazole, a potent CYP3A4 inhibitor, on the pharmacokinetics of solifenacin in healthy volunteers. In a single-site, open-label, monosequence, crossover study, 17 healthy men and women aged 18 to 65 years received a single 10 mg oral dose of solifenacin, which is is the highest available dose. After a 14-day wash-out period, they began 20 days of oral ketoconazole at a dose of 200 mg once daily. A single 10 mg dose of solifenacin was administered again on day 7 of ketoconazole treatment. Pharmacokinetics was assessed using the standard measurements of maximum plasma concentration (Cmax), time to Cmax, area under the curve (AUC), and elimination half-life (t1/2). Co-administration of ketoconazole resulted in a 1.43 times increase in the C(max) of solifenacin and an approximately 2 times increase in AUC. The mean t1/2 of solifenacin was extended from 49.3 to 77.5 hr whereas time to Cmax did not change. No substantial increase in the overall rate of adverse events, and no significant effects on vital signs, electrocardiography, clinical laboratory values, or physical examinations were noted. Administration of 200 mg ketoconazole once daily in healthy male volunteers resulted in a 2 times increase in exposure of a single 10 mg dose of solifenacin. Since ketoconazole is one of the strongest inhibitors of CYP3A4, it is expected that co-administration of other CYP3A4 inhibitors will not result in a stronger increase in solifenacin exposure.

Suc-HSA is a potent HIV-inhibitor with possible application in man. To facilitate the assessment of dosing regimens for future phase I clinical studies, we predicted the pharmacokinetic properties of Suc-HSA in man. Slices prepared from rat, monkey and human liver were incubated with succinylated albumin, and the maximum uptake rate V. and Michaelis-Menten constant K. were calculated. The pharmacokinetics after multiple doses of Suc-HSA were studied in rats. The pharmacokinetic parameters of Suc-HSA in man were predicted from the results and data from literature, using pharmacokinetic modeling and interspecies scaling techniques, and potential intravenous dose regimens for HIV treatment in man were calculated. On the basis of in vitro uptake studies in rat, monkey and human liver slices and in vivo disposition data in monkey (data from earlier study) and rat, we predicted the following parameters for liver uptake in humans: V-m 82.5 mu g h(-1) kg(-1) and K-m 0.228 mu g ml(-1). The predicted steady-state concentration after daily intravenous bolus doses of 1 mg kg(-1) is between 4 and 30 mu g ml(-1), i.e. well above the IC50 of about 0.4 mu g ml(-1). Additional loading doses of 8 mg kg(-1) in total are needed to reach steady-state within a few days. (C) 2005 Elsevier B.V. All rights reserved.

The pharmacokinetic profile of solifenacin succinate (YM905; Vesicare), a new once-daily bladder-selective muscarinic receptor antagonist, was examined in 2 controlled trials of healthy young men. A single-dose study evaluated 5-, 10-, 20-, 40-, 60-, 80-, and 100-mg doses. A multidose study evaluated 5-, 10-, 20-, and 30-mg doses. In the single-dose study, mean time to maximal concentration and elimination half-life ranged from 3.3 to 4.8 and from 40.2 to 57.6 hours, respectively; in the multidose study, the corresponding ranges were 2.9 to 5.8 and 45.0 to 64.8. Plasma concentration and area under the curve increased linearly with single doses in both trials. At steady state, a less regular increase was seen, with higher values in the 20-mg group than in the 30-mg group. All doses in the single-dose study were well tolerated. At steady state, only the 30-mg dose was not well tolerated. The most commonly reported adverse events were dry mouth, blurred vision, and headache. Solifenacin 5 and 10 mg, either as single doses or at steady state, had minimal effect on salivary flow, visual nearpoint, and the incidence of adverse events. Solifenacin was well tolerated up to single doses of 100 mg and after multiple doses of 20 mg. Its pharmacokinetic profile makes it suitable for qd administration.

Lactoferrin (Lf) is a potential drug candidate for the treatment of oropharyngeal Candida infections. However, for an effective therapeutic treatment an appropriate dosage form is required. Therefore a mucoadhesive tablet for buccal application was developed. Tablets of sufficient strength could be produced on high speed tabletting machines, but they could only be obtained when the protein contained at least 7% moisture. The tablet contained sodium alginate both for its release-controlling properties as well as for its mucoadhesive properties. Furthermore, phosphate buffer was added to keep the pH of the saliva in the mouth within the range of 6.5 to 7. 5. In this pH range, Lf has shown to have its highest activity against Candida growth inhibition. The tablet formulation containing Lf had the same antifungal properties as compared with Lf alone, because in most cases identical inhibitory concentrations were observed against several clinical isolates of Candida albicans and Candida glabrata.In human volunteers the tablets, containing 250 mg Lf and placed in each pouch, were able to keep the Lf concentration in the saliva at effective levels for at least 2 hr, while the pH of the saliva remained within the desired range. We concluded that the developed mucoadhesive tablet can improve the therapeutic efficacy of Lf and that it is suitable for further clinical research.

Although skin is the largest organ of the human body, cutaneous drug metabolism is often overlooked, and existing experimental models are insufficiently validated. This proof-of-concept study investigated phase II biotransformation of 11 test substrates in fresh full-thickness human skin explants, a model containing all skin cell types. Results show that skin explants have significant capacity for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation. Novel skin metabolites were identified, including acyl glucuronides of indomethacin and diclofenac, glucuronides of 17β-estradiol, N-acetylprocainamide, and methoxy derivatives of 4-nitrocatechol and 2,3-dihydroxynaphthalene. Measured activities for 10 μM substrate incubations spanned a 1000-fold: from the highest 4.758 pmol·mg skin(-1)·h(-1) for p-toluidine N-acetylation to the lowest 0.006 pmol·mg skin(-1)·h(-1) for 17β-estradiol 17-glucuronidation. Interindividual variability was 1.4- to 13.0-fold, the highest being 4-methylumbelliferone and diclofenac glucuronidation. Reaction rates were generally linear up to 4 hours, although 24-hour incubations enabled detection of metabolites in trace amounts. All reactions were unaffected by the inclusion of cosubstrates, and freezing of the fresh skin led to loss of glucuronidation activity. The predicted whole-skin intrinsic metabolic clearances were significantly lower compared with corresponding whole-liver intrinsic clearances, suggesting a relatively limited contribution of the skin to the body's total systemic phase II enzyme-mediated metabolic clearance. Nevertheless, the fresh full-thickness skin explants represent a suitable model to study cutaneous phase II metabolism not only in drug elimination but also in toxicity, as formation of acyl glucuronides and sulfate conjugates could play a role in skin adverse reactions.

Human aldehyde oxidase (AO) is a molybdoflavoenzyme that commonly oxidizes azaheterocycles in therapeutic drugs. Although high metabolic clearance by AO resulted in several drug failures, existing in vitro-in vivo correlations are often poor and the extrahepatic role of AO practically unknown. This study investigated enzymatic activity of AO in fresh human skin, the largest organ of the body, frequently exposed to therapeutic drugs and xenobiotics. Fresh, full-thickness human skin was obtained from 13 individual donors and assayed with two specific AO substrates: carbazeran and zoniporide. Human skin explants from all donors metabolized carbazeran to 4-hydroxycarbazeran and zoniporide to 2-oxo-zoniporide. Average rates of carbazeran and zoniporide hydroxylations were 1.301 and 0.164 pmol⋅mg skin(-1)⋅h(-1), resulting in 13 and 2% substrate turnover, respectively, after 24 hours of incubation with 10 μM substrate. Hydroxylation activities for the two substrates were significantly correlated (r(2) = 0.769), with interindividual variability ranging from 3-fold (zoniporide) to 6-fold (carbazeran). Inclusion of hydralazine, an irreversible inhibitor of AO, resulted in concentration-dependent decrease of hydroxylation activities, exceeding 90% inhibition of carbazeran 4-hydroxylation at 100 μM inhibitor. Reaction rates were linear up to 4 hours and well described by Michaelis-Menten enzyme kinetics. Comparison of carbazeran and zoniporide hydroxylation with rates of triclosan glucuronidation and sulfation and p-toluidine N-acetylation showed that cutaneous AO activity is comparable to tested phase II metabolic reactions, indicating a significant role of AO in cutaneous drug metabolism. To our best knowledge, this is the first report of AO enzymatic activity in human skin.

The absorption, distribution, metabolism, and excretion of the hedgehog pathway inhibitor sonidegib (LDE225) were determined in healthy male subjects.Six subjects received a single oral dose of 800 mg ¹⁴C-sonidegib (74 kBq, 2.0 µCi) under fasting conditions. Blood, plasma, urine, and fecal samples were collected predose, postdose in-house (days 1-22), and during 24-h visits (weekly, days 29-43; biweekly, days 57-99). Radioactivity was determined in all samples using accelerator mass spectrometry (AMS). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine concentrations of sonidegib and its main circulating metabolite in plasma. Metabolite profiles and structures were determined in pooled plasma, urine, and fecal samples using high-performance LC-AMS and LC-MS/MS, respectively.A single dose of ¹⁴C-sonidegib was well tolerated in healthy subjects. Unchanged sonidegib and total radioactivity reached peak concentration in plasma by 2 and 3 h, respectively, and demonstrated similarly long half-lives of 319 and 331 h, respectively. Absorbed sonidegib (estimated 6-7 %) was extensively distributed, and the approximate terminal volume of distribution was 2,500 L. Unchanged sonidegib and a metabolite resulting from amide hydrolysis were the major circulating components (36.4 and 15.4 % of radioactivity area under the curve, respectively). Absorbed sonidegib was eliminated predominantly through oxidative metabolism of the morpholine part and amide hydrolysis. Unabsorbed sonidegib was excreted through the feces. Metabolites in excreta accounted for 4.49 % of the dose (1.20 % in urine, 3.29 % in feces). The recovery of radioactivity in urine and feces was essentially complete (95.3 ± 1.93 % of the dose in five subjects; 56.9 % of the dose in one subject with incomplete feces collection suspected).Sonidegib exhibited low absorption, was extensively distributed, and was slowly metabolized. Elimination of absorbed sonidegib occurred largely by oxidative and hydrolytic metabolism.

Generation of early information on metabolic pathways, metabolite structures and their systemic exposure is a highly time consuming activity during the drug development process. Since these data have become of higher interest for the health authorities, efforts have been made to provide results as early as possible. ViewLux UltraHTS Microplate Imager is an instrument originally designed for high throughput in biological assays requiring luminescence, absorbance or fluorescence detections. In this work, we evaluate the capability of the new generation of the instrument for both 14C and 3H detection. We discuss data processing of the Viewlux, especially the background subtraction in comparison to conventional TopCount® instruments, as this has an impact on the limit of detection for samples containing low amounts of radioactivity like samples originating from ADME studies. We demonstrate that the limit of detection can be lowered by prolonging the exposure time for 3H labeled compounds up to 2 h. We validated the ViewLux for our metabolite profiling applications (in vitro and in vivo ADME samples) in early drug development using UPLC followed by fraction collection in 384 well plates and demonstrated for our applications that limits of detection of 2.2 and 24 dpm/well for 14C and 3H, respectively could be reached and that the throughput could be increased by at least two fold compared to conventional Topcount detection. We also demonstrate in this work how endogenous interferences resulting in false positive peaks in samples containing low amounts of radioactivity can be overcome by using a customized light filter.

SBR759 is a novel polynuclear iron(III) oxide-hydroxide starch·sucrose·carbonate complex being developed for oral use in chronic kidney disease (CKD) patients with hyperphosphatemia on hemodialysis. SBR759 binds inorganic phosphate released by food uptake and digestion in the gastro-intestinal tract increasing the fecal excretion of phosphate with concomitant reduction of serum phosphate concentrations. Considering the high content of ∼20% w/w covalently bound iron in SBR759 and expected chronic administration to patients, absorption of small amounts of iron released from the drug substance could result in potential iron overload and toxicity. In a mechanistic iron uptake study, 12 healthy male subjects (receiving comparable low phosphorus-containing meal typical for CKD patients: ≤1000 mg phosphate per day) were treated with 12 g (divided in 3 × 4 g) of stable (58)Fe isotope-labeled SBR759. The ferrokinetics of [(58)Fe]SBR759-related total iron was followed in blood (over 3 weeks) and in plasma (over 26 hours) by analyzing with high precision the isotope ratios of the natural iron isotopes (58)Fe, (57)Fe, (56)Fe and (54)Fe by multi-collector inductively coupled mass spectrometry (MC-ICP-MS). Three weeks following dosing, the subjects cumulatively absorbed on average 7.8 ± 3.2 mg (3.8-13.9 mg) iron corresponding to 0.30 ± 0.12% (0.15-0.54%) SBR759-related iron which amounts to approx. 5-fold the basal daily iron absorption of 1-2 mg in humans. SBR759 was well-tolerated and there was no serious adverse event and no clinically significant changes in the iron indices hemoglobin, hematocrit, ferritin concentration and transferrin saturation.

Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti-HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various polypeptides: proteins with a high content of basic amino acids in which all of the available epsilonNH2 groups were anionized yielded the most potent anti-HIV compounds. It remained unclear however whether the total net negative charge of the various derivatized proteins, or rather the charge density on the protein backbone, is essential for the observed anti-HIV activity. Earlier studies have shown that acylated albumins preferentially block the process of HIV/cell fusion through binding to the HIV envelope proteins gp120 and gp41 as well as to the cell surface of the HIV target cells. Some of these polyanionic proteins have been shown to interfere also with the gp120-CD4 mediated virus/cell binding. The relative contribution of these effects to the anti-HIV activity may depend both on the total negative charge introduced as well as the hydrophobicity of the acylating reagent added to the particular proteins. In this study we show that the higher the charge density of the derivatized proteins, the more potent their HIV replication inhibiting effects are. In contrast, the addition of positive charge to the studied plasma and milk proteins through amination resulted in a reduced anti-HIV activity but a clearly increased anti-HCMV activity, with IC50 values in the low micromolar concentration range. Interestingly, native lactoferrin (Lf) was antivirally active against both HIV and HCMV. Acylation or amination of Lf increased the anti-HIV and anti-HCMV activity, respectively. The N-terminal portion of Lf appeared essential for its anti-HCMV effect: N-terminal deletion variants of human Lf were less active against HCMV. Circular dichroism of the modified proteins showed that the secondary structure of the tested proteins was only moderately influenced by acylation and/or covalent attachment of drugs, making these (derivatized) proteins useful candidates as antiviral agents and/or intrinsically active drug carriers. The relatively simple chemical derivatization as well as the abundant sources of blood plasma and milk proteins provides attractive opportunities for the preparation of potent and relatively cheap antiviral agents for systemic or local applications.

Because of the rising incidence of failures in the treatment of oropharyngeal candidosis in the case of severely immunosuppressed patients (mostly human immunodeficiency virus [HIV]-infected patients), there is need for the development of new, more effective agents and/or compounds that support the activity of the common antifungal agents. Since lactoferrin is one of the nonspecific host defense factors present in saliva that exhibit antifungal activity, we studied the antifungal effects of human, bovine, and iron-depleted lactoferrin in combination with fluconazole, amphotericin B, and 5-fluorocytosine in vitro against clinical isolates of Candida species. Distinct antifungal activities of lactoferrin were observed against clinical isolates of Candida . The MICs generally were determined to be in the range of 0.5 to 100 mg · ml −1 . Interestingly, in the combination experiments we observed pronounced cooperative activity against the growth of Candida by using lactoferrin and the three antifungals tested. Only in a limited concentration range was minor antagonism detected. The use of lactoferrin and fluconazole appeared to be the most successful combination. Significant reductions in the minimal effective concentrations of fluconazole were found when it was combined with a relatively low lactoferrin concentration (1 mg/ml). Such combinations still resulted in complete growth inhibition, while synergy of up to 50% against several Candida species was observed. It is concluded that the combined use of lactoferrin and antifungals against severe infections with Candida is an attractive therapeutic option. Since fluconazole-resistant Candida species have frequently been reported, especially in HIV-infected patients, the addition of lactoferrin to the existing fluconazole therapy could postpone the occurrence of species resistance against fluconazole. Clinical studies to further elucidate the potential utility of this combination therapy have been initiated.

The present study shows the lymphatic distribution of the negatively charged anti-HIV-1 agents succinylated or aconytilated human serum albumins (HSAs) in rats. Quantitation of blood and lymphatic concentrations of these proteins was performed through fluorescence detection of the fluorescein isothiocyanate (FITC)-labeled proteins. At several time points after i.v. injection, samples were taken from the cannulated thoracic duct and the carotid artery. Distribution of the negatively charged albumins (NCAs) to lymph was much more rapid than that of albumin itself and was dependent on the total net negative charge added to the protein: the half-life times of lymphatic equilibration were 15, 30, and 120 min for FITC-labeled aconytilated HSA, FITC-labeled succinylated HSA, and FITC-labeled HSA, respectively. Lymph to blood concentration ratios of the studied compounds obtained at steady state approached unity. In addition, the fluorescence in both body fluids was shown to represent unchanged labeled proteins. It was therefore inferred that the NCAs efficiently passed the endothelial barrier from blood to the interstitial compartment. Subsequently, we studied whether a specialized process was involved in the endothelial passage of the NCAs to the lymph. The following observations supported such a mechanism: a) preinjection of the scavenger receptor blockers polyinosinic- and formaldehyde-treated HSA reduced the transport from blood to the lymphatic compartment of FITC-labeled aconytilated HSA by more than 90%; b) the rate of lymphatic distribution was largely reduced when the body temperature of the rat was lowered to 28 degrees; and c) pre-administration of chloroquine resulted in a significant reduction in the lymphatic distribution of the NCAs. These data collectively indicate that a scavenger receptor-mediated process is involved in the transendothelial transport of NCAs. In situ localization in lymph nodes of the rat showed that FITC-labeled aconytilated and succinylated HSA are mainly present in the germinal center and parafollicular zones. The efficient distribution of these anionized proteins to the lymphatic system is of particular interest for HIV therapy, taking into account that replication of HIV mainly takes place in the lymphoid system. The observation that macromolecules, through charge modification, can extravasate through a receptor-mediated transcytotic process is potentially of major importance for the delivery of drugs with macromolecular carriers to cells not directly in contact with the blood.

The disposition and biotransformation of (14)C-radiolabeled mavoglurant were investigated in four healthy male subjects after a single oral dose of 200 mg. Blood, plasma, urine, and feces collected over 7 days were analyzed for total radioactivity, mavoglurant was quantified in plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and metabolite profiles were generated in plasma and excreta by high-performance liquid chromatography (HPLC) and radioactivity detection. The chemical structures of mavoglurant metabolites were characterized by LC-MS/MS, wet-chemical and enzymatic methods, NMR spectroscopy, and comparison with reference compounds. Mavoglurant was safe and well tolerated in this study population. Mavoglurant absorption was ≥50% of dose reaching mean plasma Cmax values of 140 ng/ml (mavoglurant) and 855 ng-eq/ml (total radioactivity) at 2.5 and 3.6 hours, respectively. Thereafter, mavoglurant and total radioactivity concentrations declined with mean apparent half-lives of 12 and 18 hours, respectively. The elimination of mavoglurant occurred predominantly by oxidative metabolism involving primarily 1) oxidation of the tolyl-methyl group to a benzyl-alcohol metabolite (M7) and subsequently to a benzoic acid metabolite (M6), and 2) oxidation of the phenyl-ring leading to a hydroxylated metabolite (M3). The subjects were mainly exposed to mavoglurant and seven main metabolites, which combined accounted for 60% of (14)C-AUC0-72 h (area under the concentration-time curve from time 0 to infinity). The primary steps of mavoglurant metabolism observed in vivo could partially be reproduced in vitro in incubations with human liver microsomes and recombinant cytochrome P450 enzymes. After 7 days, the mean balance of total radioactivity excretion was almost complete (95.3% of dose) with 36.7% recovered in urine and 58.6% in feces.

A number of native and modified milk proteins from bovine or human sources were analyzed for their inhibitory effects on human immunodeficiency virus type 1 (HIV-1) and HIV-2 in vitro in an MT4 cell test system, The proteins investigated were lactoferrin, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B.By acylation of the amino function of the lysine residues in the proteins, using anhydrides of succinic acid or cis-aconitic acid, protein derivatives were obtained that all showed a strong antiviral activity against human immunodeficiency virus type 1 and/or 2. The in vitro IC50 values of the aconitylated proteins were in the concentration range of 0.3 to 3 nM. Succinylation or aconitylation of alpha-lactalbumin and beta-lactoglobulin A/B also produced strong anti-HIV-2 activity with IC50 values on the order 500 to 3000 nM. All compounds showed virtually no cytotoxicity at the concentration used.Peptide-scanning studies indicated that the native lactoferrin as well as the charged modified proteins strongly bind to the V3 loop of the gp120 envelope protein, with K-d values in the same concentration range as the above-mentioned IC50. Therefore, shielding of this domain, resulting in inhibition of virus-cell fusion and entry of the virus into MT4 cells, may be the likely underlying mechanism of antiviral action.

Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using (14)C or (3)H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector.

The pharmacokinetics of the dopamine D2 agonist S(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (1, N-0923) after intravenous, intraperitoneal, and oral administration was studied in freely moving male and female albino Wistar rats. In all cases, the dose was 1 0 mumol . kg-1. Levels of 1 in plasma were monitored up to 6 h after dosing by high-performance liquid chromatography. A biphasic disappearance pattern with a rapid distribution phase was observed in the curve of concentration in plasma versus time. Pharmacokinetic analysis based on a two-compartment model with elimination from the central compartment yielded the following parameters for male rats (mean +/-standard deviation, n = 3): elimination half-life was 108 +/- 7 min, apparent volume of distribution of the central compartment was 397 +/- 44 mL . kg-1, the plasma clearance was 32 +/- 4 mL . min-1 . kg-1, and the apparent volume of distribution at steady state was 2.29 +/- 0.25 L . kg-1. No significant difference (analysis of variance p value > 0.25) was found in the pharmacokinetic data between male and female rats. An extremely fast absorption was found after intraperitoneal administration. Maximal concentrations in plasma were often observed at the first time point 5 min after dosing. The bioavailability (mean +/- standard deviation, n = 3) was 7.9 +/- 2.7% for male rats and 6.5 +/- 2.1% for female rats. An increase of the elimination half-life of at least 40% for male rats and 300% for female rats was observed, indicating dose-dependent kinetics after intraperitoneal administration. After oral dosing, levels of 1 in plasma were extremely low and often below the quantitation limit of the assay (10 pmol . mL-1). This did not allow detailed kinetic analysis. Even in the more favorable cases, the bioavailability did not exceed 0.5%. Although the 5-0-glucuronide and 5-sulfate are the major metabolites of 1, they could not be detected in plasma, indicating efficient excretion into bile and urine.

An innovative custom-built atmospheric ionization source afforded an opportunity to perform on-line LC/MS analysis and to obtain identification of metabolites without need to rely on radioactive profiling. An HPLC with a UV detector coupled to a modified R 3010 triple quadrupole mass spectrometer was used in which nitrogen gas effectively prevented the clustering of ions with polar solvent molecules as well as acting as the nebulizing gas for the ionspray LC/MS interface. This approach was useful in elucidating the oxidative metabolism of the potent dopamine D2 agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin in rat-, monkey-, and human liver microsome preparations. Microsomes from monkey showed the highest metabolic activity, with 8.3 nmol · min−1 · mg−1 protein; the human material resulted in the lowest, 1.2 nmol · min−1 · mg−1 protein. Metabolic profiling showed that the three species produced the same metabolites, but to different extents. Liver microsomes metabolized the (-) enantiomer to...

The penetration of the dopaminergic D2 agonist S (−)-2-( N -propyl- N -2-thienylethylamino)-5-hydroxytetralin (N-0923) was evaluated in vitro on full-thickness rat skin, using the Franz diffusion cell. The drug was tested as the N-0923 · HCl salt as well as the N-0923 base. The penetration-enhancing effect of Azone was studied in a vehicle concentration of 5%, or after pretreating shaven rat skin in vivo, at several time intervals before the in vitro experiment, with a solution containing 1 or 5% Azone. From the vehicle containing 60% ethanol, 20% propylene glycol and 20% water, the extent of penetration through rat skin was relatively low for the base and even lower for the salt. The introduction of Azone into the vehicle in a final concentration of 5% resulted in a 6-fold decrease in penetration for the free base. Nevertheless, penetration of the salt increased about 12-fold in extent during the first 12 h, after which it declined to almost zero. The lag time was reduced from 13 to 5 h. Pretreatment of rat skin in vivo with an ethanol-propylene glycol-water solution containing 5% Azone resulted in a 12-fold increase in flux for the salt, a 2-fold increase in flux for the base and a decrease in lag time to about the same values for both N-0923 · HCl and N-0923 base. In vivo pretreatment of rat skin with a solution containing 1% Azone had no significant effect on the flux and lag time vs controls. This study indicates that Azone may have potential in facilitating the transdermal application of N-0923, principally through pretreatment of the skin to enhance absorption. This may allow it to circumvent its considerable oral first-pass metabolism.

This chapter contains sections titled: Frequency Analysis of Non-Sinusoidal Waveforms: Practical Considerations; Analog-to-Digital Conversion; Sequence Component Analysis; IEEE 1459: Power Definitions for modern Power Systems; Localization of Sources of Waveform Distortion in a Modern Power System; Bibliography

Background: Solifenacin succinate (YM905; Vesicare®, Astellas Pharma Inc., Tokyo, Japan) is a new once-daily, orally administered muscarinic receptor antagonist under investigation for the treatment of overactive bladder. Objective: The aim of this study was to evaluate the effect of solifenacin on the pharmacokinetic (PK) parameters of an oral contraceptive (OC) containing ethinyl estradiol (EE) and levonorgestrel (LNG). Methods: In a double-blind, placebo-controlled, 2-period, crossover study, 24 healthy, young, white women received a combined OC (EE 30 μg + LNG 150 μg) daily for two 21-day cycles, separated by a 7-day washout. On day 12 of each cycle, subjects began a 10-day regimen of solifenacin 10 mg QD, which is 2 times the suggested starting dose, or placebo. Subjects crossed over to the other treatment arm for the second cycle. Primary PK end points were C max and AUC from time 0 to 24 hours (AUC 0–24h ) for EE and LNG. Women ranged in age from 20 to 37 years and had a mean body weight of 64 kg, mean height of 167.4 cm, and mean body mass index of 23 kg/m 2 . Seven women had never smoked, while 5 were former smokers and 12 were regular smokers. Safety assessments included the nature, frequency, and severity of spontaneously reported or observed adverse events, vital signs, electrocardiogram, laboratory values, and physical examination. Results: Statistical analysis of AUC 0–24h /product of baseline concentration and total blood sampling time, and C max /baseline concentration ratios of solifenacin versus placebo for EE and LNG found the 90% CI to be within the predefined range of 0.8 to 1.25 (EE: 0.854–1.164 and 0.822–1.167; LNG: 0.920–1.125 and 0.910–1.139). The number of samples with non-quantifiable luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels were comparable after administration of the OC with either solifenacin or placebo. The adverse event most frequently reported was dry mouth (solifenacin, n=25 [9 mild, 13 moderate, and 3 severe] vs placebo, n=1 [moderate]). There were no clinically relevant effects on vital signs, electrocardiogram, or laboratory parameters. Conclusions: A PK interaction between solifenacin and the OC containing EE and LNG was not found in this study. Solifenacin was not found to have altered suppression of LH or FSH. The drug was well tolerated in these healthy, young, white, adult female volunteers.

Lactoferrin, an iron-binding glycoprotein, is a potential agent for the treatment of oropharyngeal Candidiasis. The aim of the present study was to test the capability of lactoferrin, combined or not combined with conventional antifungal agents, to inhibit the growth of different Candida species under various experimental conditions to be of guidance in the development of a suitable pharmaceutical formulation containing lactoferrin. The anti-Candida activities of lactoferrin were considerably higher using RPMI instead of SLM as assay medium. They were moreover increased by raising the medium pH from 5.6 to 7.5. With the 'standard' antifungal agent fluconazole similar results were found as for lactoferrin, but the medium type and pH did not affect MIC values of amphotericin B. The addition of saliva to medium did not reduce the antifungal activities of the individual compounds. Synergistic inhibitory effects on Candida growth were found for combinations of lactoferrin and fluconazole or amphotericin B, irrespective of the medium type and pH, or the addition of saliva. This indicates that for treatment of oral Candidiasis a formulation containing lactoferrin seems appropriate; results may be optimized if the formulation is provided with buffer capacity to attain pH 7.5 in the mucosal fluid. The synergistic effects between lactoferrin and 'standard' antifungals indicate that combinations should be considered in such a formulation.

The pharmacokinetics and metabolic fate of the intrinsically active (anti-HIV) drug carrier succinylated human serum albumin (Suc-HSA) was studied in rats. Suc-HSA was prepared by derivatizing HSA with 1,4-[C-14]-succinic anhydride, a modification by which all available epsilonNH2-groups in HSA were converted into carboxylic groups.After iv injections of 0.3, 1.0, 3.0 and 10.0 mg/kg in freely moving rats, Suc-HSA showed a dose dependent elimination pattern, indicating a saturable elimination pathway. The Michaelis-Menten parameters V-max and K-m were 98.7 mug.min(-1).kg(-1) and 8.5 mug.ml(-1) respectively. The kinetics of Suc-HSA was influenced by anaesthesia. In anaesthetised animals, V-max and K-m were found to be 26.9 mug.min(-1).kg(-1) and 0.26 mug.ml(-1), respectively. This implies an intrinsic clearance of 100 ml.min(-1).kg(-1), which is about 10-fold higher as compared to 12 ml.min(-1).kg(-1) in freely moving animals.Intravenous administration of a sub-saturable dose of 3.0 mg.kg(-1)1,4-[C-14]-Suc-HSA to freely moving rats resulted in a biphasic elimination with an initial t(1/2) of 20 min and a terminal t(1/2) of 40 hrs. Excretion of metabolites in urine and faeces lasted for at least 48 hours. About 70% of the radioactive dose was excreted in urine, whereas maximally 2% was detected in faeces. Suc-HSA was degraded to its individual amino acids including succinylated lysine (the only radioactive product formed). Succinylated lysine was not further metabolised and mainly excreted via the urine. Immunohistochemical staining showed that even after 48 hrs Suc-HSA could be detected in livers. Together with the urinary excretion patterns, this points to a gradual degradation of Suc-HSA.

In the present study we investigated the applicability of the liver slice model to study mechanisms of drug uptake. Four model compounds were investigated that enter hepatocytes via entirely different membrane transport mechanisms. Rhodamine B (RB), which enters hepatocytes by passive diffusion, was homogeneously distributed throughout the rat liver slice (250 mum thickness) within 5 min, indicating that the penetration rate into the slice and the diffusion rate into the cells are rapid. In contrast, lucigenin (LU), which is taken up by hepatocytes through adsorptive endocytosis, was detected in the inner cell layers after 15 min. Digoxin uptake into the slice showed a temperature-dependent component and was stereos electively inhibited by quinine, which is compatible with the involvement of a carrier-mediated uptake mechanism. The neo-glycoalbumin Lactose(27)-Human Serum Albumin (Lact(27)-HSA) and the negatively charged Succinylated-Human Serum Albumin (Suc-HSA) entered the slices and were taken up temperature-dependently into hepatocytes and endothelial cells, respectively. The liver slice preparation is a valuable tool to investigate the mechanisms of cellular uptake of drugs. Moreover, the precision-cut liver slices offer the unique possibility to study both hepatocyte and endothelial cell function in human and rat liver. (C) 2001 Elsevier Science Inc. All rights reserved.

The determination of the enantiomeric impurity, i.e, the percentage of (+) N-0437 (= N-0924) in several batches of (-) N-0437 (= N-0923) by chiral HPLC is described. Enantiomeric impurities were calculated based on the peak areas of the two baseline separated enantiomers in the chromatogram. The enantiomeric impurities found in different batches ranged from 0.02% to 0.11%. Calibration curves of the two isomers of N-0437 (Fig. 1,) were made twice to study the reproducibility and linearity of the method. The absorbance ratio, N-0923/N-0924, was found to be 1.02 with a relative standard deviation (RSD) of 9% over the whole concentration range used for the calibration curves.

Negatively charged albumins (NCAs) have been identified as potent inhibitors of HIV-1 replication in vitro. Time of addition studies suggest that succinylated and aconitylated human serum albumin (Suc-HSA and Aco-HSA) act at an early stage of the virus life cycle, and surface plasmon resonance (BIAcore) experiments have confirmed a direct interaction of NCAs with HIV-1 gp120, Resistance to Suc-HSA and Aco-HSA was analyzed by characterizing HIV-1 variants that were selected in cell culture after serial passage of the NL4-3 strain in the presence of the compounds. After 24 passages (126 days) we isolated variants that were resistant to Suc-HSA (>27-fold) and Aco-HSA (37-fold), as compared with the wild-type NL4-3 virus. The binding of the NCA-resistant HIV strains to CD4(+) MT-4 cells could no longer be inhibited by either Suc- or Aco-HSA, The emergence of mutations in the envelope gp120 of the resistant virus paralleled the emergence of the resistant phenotype, The Suc-HSA-resistant strain was 100-fold cross-resistant to the G quartet-containing oligonucleotide AR177 (Zintevir, an HIV-binding inhibitor), and partially cross-resistant to dextran sulfate, but remained sensitive to the bicyclam AMD3100 and the chemokine SDF-1 alpha, which block HIV replication by interaction with the chemokine receptor CXCR4, Furthermore, neither Suc-HSA nor Aco-HSA inhibited the binding of monoclonal antibodies 12G5 and 2D7 (directed to CXCR4 and CCR5, respectively) in SUPT-1 cells or THP-1 cells. These results confirm that NCAs bind primarily to gp120 and do not interact directly with the HIV chemokine receptor but block the binding of the virus particles (through gp120) with CD4(+) cells.

Succinylated human serum albumin (Suc-HSA) was synthesized by treating human serum albumin with succinic anhydride, Among similar proteins and neo(glyco)proteins tested, Suc-HSA exhibits a pronounced net negative charge, a feature that largely contributes to its efficacy against replication of human immunodeficiency virus type 1 (HIV-1), To assess further the antiviral effect of Suc-HSA, the effect on HIV-1 replication was studied in the presence of whole human plasma, Pretreatment of MT2 cells with Suc-HSA was more efficacious than direct Suc-HSA treatment of HIV prior to addition to the cells, No changes in the antiviral effect of Suc-HSA were observed in tissue culture medium, 30% plasma, or whole plasma when CPDA-1 (citrate-phosphate-dextrose-adenine 1) was used as the anticoagulant, However, a dramatic decrease (greater than 99%) in the antiviral activity was observed when these experiments were performed in plasma prepared from blood using heparin as anticoagulant, The antagonistic effect by heparin was observed both in the case that heparin was added prior to or after addition of Suc-HSA to the test system, In the present study we demonstrate that heparin largely reduces Suc-HSA activity on HIV replication in the same concentration in which if affects binding of Suc-HSA to the envelope protein gp120 and in particular its V3 domain, In the same concentration range, heparin reduced binding of Suc-HSA to MT4 cells, another HTLV-I-transformed cell line, It is concluded that heparin can displace Suc-HSA from its binding sites on hybrid lymphoid cells as well as on HIV-1 particles, Therefore, we conclude that both the binding to cells and to virus contribute to the potent anti-HIV-1 effect, The fact that heparin and heparin degradation products antagonize Suc-HSA without having a significant anti-HIV-1 effect indicates that the anticoagulant acts as a relatively weak partial inhibitor.

We previously reported the antiviral capacity of human serum albumin (HSA), which was modified by the introduction of a single (Suc-HSA) or two carboxylic groups (Aco-HSA) per lysine residue, yielding strongly negatively charged polypeptides. Here we report the antiviral effect of these modified HSAs on replication of primary HIV-1 isolates that differed with respect to syncytium-inducing (SI) capacity and cell tropism. Both Suc-HSA and Aco-HSA potently inhibited replication of primary HIV-1 variants, independent of the SI capacity of the HIV-1 variant, with IC50 values in the range of 50 to 187 microg/ml. The inhibition of the formation of syncytia and the absence of proviral DNA products in cells inoculated with HIV-1 in the presence of Suc-HSA or Aco-HSA pointed to interference at an early level in the virus replication cycle. The inhibitory capacity of Suc-HSA and Aco-HSA on primary HIV-1 variants suggests that these agents are potential candidates for use in antiviral therapy in HIV-infected individuals.

Negatively charged albumins (NCAs, with the prototypes succinylated human serum albumin (Suc-HSA) and aconitylated human serum albumin (Aco-HSA)), modified proteins with a potent anti-human immunodeficiency virus type 1 (anti-HIV-1) activity in vitro, were studied for their pharmacokinetic behaviour in mice and their in vivo anti-HIV-1 efficacy in an HIV-1 infection model in mice. In contrast to the saturation kinetics found in rats, intravenous injections of 10-300 mg/kg for both NCAs showed a linear correlation between the area under the curve (AUC) and the dose. The elimination t1/2 was 25 and 30 min for Suc-HSA and Aco-HSA, respectively. Preinjections of an excess of formaldehyde-treated albumin (Form-HSA) resulted in plasma levels that were 3- and 4-fold higher for Aco-HSA and Suc-HSA, respectively. These data indicate that elimination is at least partly (scavenger) receptor-mediated. Organ distribution studies 10 min after injection showed an accumulation in liver (Suc-HSA 17.3 +/- 6.6% of the dose; Aco-HSA 20.9 +/- 2.3%) and lungs (Suc-HSA 12.7 +/- 10.5%; Aco-HSA 16.0 +/- 13.6). Intraperitoneal injection of 300 mg/kg Suc-HSA resulted in a final bioavailability of about 0.45. Suc-HSA was also evaluated for its in vivo neutralizing capacity in a human-to-mouse chimeric model for HIV-1 infection. Intraperitoneal injections of 300 and 3 mg/kg Suc-HSA, given 15-30 min before the mice were challenged with the virus, sufficed to protect these mice against infection with the HIV-1 IIIB strain.

Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

The metabolism of the dopamine D2 agonist N-0923 was investigated by an in vitro isolated liver perfusion. Determining the metabolic profile and identity of the different metabolites was achieved by using high-performance liquid chromatography with UV detection, combined with atmospheric pressure ionization mass spectrometry. Using this technique, no extensive sample cleanup is required, and the studies can be performed without radioactivity. In addition to previously observed metabolites, nine new metabolic products were identified. All metabolites were exclusively excreted into the bile, except for the despropyl metabolite, which was also detectable in the perfusate. 5-O-Glucuronidation and N-depropylation followed by 5-O-glucuronidation are the most important metabolic routes. N-dealkylation of the thienylethyl group followed by 5-O-glucuronidation and sulfation is a second major metabolic pathway. Catechol formation of the despropyl metabolite with or without subsequent conjugation was not found. Catechol formation of the desthienylethyl metabolite occurred, but only its glucuronide conjugates were found. This study complements previous results of in vivo metabolic studies using the radiolabeled racemate N-0437, and it explains differences in bile excretion during isolated liver perfusions using N-0923 and radiolabeled N-0923.

The transdermal penetration of the dopamine D2 agonist, N-0923, was studied in freely moving male albino Wistar rats, after cutaneous dosing of 2 μmol cm −2 in a carbomer gel. A silicone patch with an effective area of 0.65 or 1.3 cm 2 was filled with 100 or 200 mg gel, respectively. The patch was attached to the rat skin using histoacrylic glue. The drug was tested as N-0923 · HCl. The enhancing effect of azone was studied by adding 5% of the penetration enhancer to the gel. A second enhancement study was performed after pretreatment of shaved rat skin with a solution containing 5% azone at various time points, prior to administrations of drug. The pretreatment solution contained ethanol-propyleneglycol-water-azone (60 : 20 : 15 : 5 v/v). The dermal penetration of N-0923 · HCl from the carbomer gel resulted in plasma levels which were relatively low and often at or just above the limit of quantitation of the assay (10 pmol ml −1 ). Adding 5% azone to the gel resulted in undetectable plasma levels, indicating the drug not being released from the carrier. Pretreatment of the rat skin with a solution containing 5% azone resulted in a 3-fold increase in the flux vs controls. Plasma levels were detectable up to 10 h after application. All pretreated animals showed stereotypy, indicating that adequate amounts of the drug reached the brain.

The in vitro glucuronidation of seven monohydroxy-2-aminotetralins and two naphthoxazines has been determined using human and rat liver microsomes. All these compounds stimulate the D2 dopamine receptor. The influence of the position of the phenolic hydroxyl group was studied with rat microsomes in monohydroxy-2-(N,N-dipropylamino)-tretralins. The highest activity and intrinsic clearance was found for 7-OH-DPAT, but the latter values for 5-OH-DPAT and 6-OH-DPAT were much lower by a factor of 9 and 30, respectively. The 8-OH-isomer was not glucuronidated at all. Substitution of a propyl side chain by a thienylethyl-, or phenylethyl side chain, in 5-hydroxy-DPAT, or in (+)-4-propyl-9-hydroxyhexahydronaphthoxazine (PHNO, N-0500), showed a large increase of the UDPGT affinity and intrinsic clearance especially for N-0437. It also resulted for N-0437 in a much higher affinity towards the dopaminergic D2 receptor. Although the glucuronidation activity of human microsomes was found to be considerably lower than that of rat microsomes, the latter phenomenon was clearly visible with human microsomes as well. These findings may have serious implications for the ability of these drugs to adequately reach the brain.

Two series of pUB110-derived plasmids were constructed to study segregational stability in Bacillus subtilis. pEB plasmids were based on the entire pUB110, whereas pLB plasmids lack the membrane-binding areas BA3 and BA4. Two kinds of stability defects were observed. The first was characterized by a strong size dependency and occurred with different inserts at various positions in pLB and pEB plasmids. Size-dependent reductions in plasmid copy numbers appeared to underly this phenomenon. This may render pUB110 unsuitable for the cloning of inserts larger than about 3 kb, in particular if no selective conditions can be applied. The second defect, observed with pLB plasmids, was caused by the absence of the membrane-binding areas BA3 and BA4. Deletion of BA3 resulted in the accumulation of single-stranded plasmid DNA, suggesting that BA3 contains the initiation signal for complementary strand synthesis. The BA3 region is very rich in hyphenated dyad symmetry which, in single-stranded DNA, could result in several stable alternative secondary structures. It is speculated that the activity of the BA3-associated initiation signal contributes to the segregational stability of pUB110-derived plasmids in B. subtilis. The absence of the BA3 stability function could not account for all stability defects observed. Additional stability functions seemed to be located on the BA4 fragment.

Human serum albumin (HSA) derivatized with cis-aconitic anhydride (Aco-HSA) that was earlier shown to inhibit replication of human immunodeficiency virus type 1 (HIV-1), was covalently coupled to conventional liposomes, consisting of phosphatidylcholine, cholesterol and maleimido-4-(p-phenylbutyryl)phosphatidylethanolamine, using the heterobifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA). The amount of HSA that could be coupled to the liposomes depended on derivatization of the HSA and ranged from 64.2 +/- microgram HSA/micromol total lipid for native HSA to 29.5 +/- 2.7 microgram HSA/micromol total lipid for HSA in which 53 of the epsilon amino groups of lysine were derivatized with cis-aconitic anhydride (Aco53-HSA). Incorporation of 3.8 mol% of total lipid of a poly(ethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) in the liposomes resulted in a lower coupling efficiency of Aco-HSA. The elimination and distribution of the liposomal conjugates in rats in vivo was largely dependent on the modification of the HSA coupled to the liposomes. With native HSA-liposomes, more than 70% of the conjugate was still found in the blood plasma 30 min after i.v. injection in rats, while at this time Aco-HSA-liposomes were completely cleared from the circulation. The rapid clearance of conventional Aco-HSA-liposomes was due to a rapid uptake into the liver and could be considerably decreased by incorporating PEG-PE in the liposomal bilayer. After 3 h 60% of Aco-HSA-PEG-liposome conjugates were found in the blood. In an in vitro anti-HIV-1 assay, the 50% inhibitory concentrations (IC50) for Aco39-HSA-liposomes and Aco53-HSA-liposomes expressed as protein weight, were 2.87 microgram/ml and 0.154 microgram/ml, respectively. When PEG-PE was incorporated, the Aco53-HSA-liposomes retained anti HIV-1 activity (IC50:3.13 microgram/ml). The possibility to modulate the residence time in the bloodstream of Aco-HSA-liposomes and the potent anti-HIV-1 activity of these conjugates, may allow the development of an intrinsically active drug carrier system. By incorporating anti HIV-1 drugs such as AZT into such liposomes a drug delivery system can be designed that might act simultaneously on the virus/cell binding by virtue of the coupled Aco-HSA and on the RNA/DNA transcription of the HIV-1 replication cycle through the nucleoside analogue.