A new oil/membrane approach for integrated sweat sampling and sensing: sample volumes reduced from µL's to nL's and reduction of analyte contamination from skin.

Wearable sweat biosensensing technology has dominantly relied on techniques which place planar-sensors or fluid-capture materials directly onto the skin surface. This 'on-skin' approach can result in sample volumes in the µL regime, due to the roughness of skin and/or due to the presence of hair. Not only does this increase the required sampling time to 10's of minutes or more, but it also increases the time that sweat spends on skin and therefore increases the amount of analyte contamination coming from the skin surface. Reported here is a first demonstration of a new paradigm in sweat sampling and sensing, where sample volumes are reduced from the µL's to nL's regime, and where analyte contamination from skin is reduced or even eliminated. A micro-porous membrane is constructed such that it is porous to sweat only. To complete a working device, first placed onto skin is a cosmetic-grade oil, secondly this membrane, and thirdly the sensors. As a result, spreading of sweat is isolated to only regions above the sweat glands before it reaches the sensors. Best case sampling intervals are on the order of several minutes, and the majority of hydrophilic (low oil solubility) contaminants from the skin surface are blocked. In vitro validation of this new approach is performed with an improved artificial skin including human hair. In vivo tests show strikingly consistent results, and reveal that the oil/membrane is robust enough to even allow horizontal sliding of a sensor.

The microfluidics of the eccrine sweat gland, including biomarker partitioning, transport, and biosensing implications.

Non-invasive and accurate access of biomarkers remains a holy grail of the biomedical community. Human eccrine sweat is a surprisingly biomarker-rich fluid which is gaining increasing attention. This is especially true in applications of continuous bio-monitoring where other biofluids prove more challenging, if not impossible. However, much confusion on the topic exists as the microfluidics of the eccrine sweat gland has never been comprehensively presented and models of biomarker partitioning into sweat are either underdeveloped and/or highly scattered across literature. Reported here are microfluidic models for eccrine sweat generation and flow which are coupled with review of blood-to-sweat biomarker partition pathways, therefore providing insights such as how biomarker concentration changes with sweat flow rate. Additionally, it is shown that both flow rate and biomarker diffusion determine the effective sampling rate of biomarkers at the skin surface (chronological resolution). The discussion covers a broad class of biomarkers including ions (Na(+), Cl(-), K(+), NH4 (+)), small molecules (ethanol, cortisol, urea, and lactate), and even peptides or small proteins (neuropeptides and cytokines). The models are not meant to be exhaustive for all biomarkers, yet collectively serve as a foundational guide for further development of sweat-based diagnostics and for those beginning exploration of new biomarker opportunities in sweat.

Vitamin B12-responsive severe leukoencephalopathy and autonomic dysfunction in a patient with "normal" serum B12 levels.

Leukoencephalopathy and autonomic dysfunction have been described in individuals with very low serum B(12) levels (<200 pg/ml), in addition to psychiatric changes, neuropathy, dementia and subacute combined degeneration. Elevated homocysteine and methylmalonic acid levels are considered more sensitive and specific for evaluating truly functional B(12) deficiency. A previously healthy 62-year-old woman developed depression and cognitive deficits with autonomic dysfunction that progressed over the course of 5 years. The patient had progressive, severe leukoencephalopathy on multiple MRI scans over 5 years. Serum B(12) levels ranged from 267 to 447 pg/ml. Homocysteine and methylmalonic acid levels were normal. Testing for antibody to intrinsic factor was positive, consistent with pernicious anaemia. After treatment with intramuscular B(12) injections (1000 µg daily for 1 week, weekly for 6 weeks, then monthly), she made a remarkable clinical recovery but remained amnesic for major events of the last 5 years. Repeat MRI showed partial resolution of white matter changes. Serum B(12), homocysteine and methylmalonic acid levels are unreliable predictors of B(12)-responsive neurologic disorders, and should be thoroughly investigated and presumptively treated in patients with unexplained leukoencephalopathy because even long-standing deficits may be reversible.

Optimization of naphthalimide-imidazoacridone with potent antitumor activity leading to clinical candidate (HKH40A, RTA 502).

Unsymmetrical bifunctional antitumor agent WMC79 was further optimized to generate compound 7b that not only inhibited the growth of many tumor cell lines, but caused rapid apoptosis. Unlike the parent compound, 7b is toxic to both p53 positive and negative cancer cells. It has potent in vivo activity against xenografts of human colon and pancreatic tumors in athymic mice.

Anti-tumor drug candidate 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole induces single-strand breaks and DNA-protein cross-links in sensitive MCF-7 breast cancer cells.

PURPOSE: The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) exhibits selective and potent anticancer activity, and its lysylamide prodrug (Phortress, NSC 710305) recently entered Phase I clinical trials in the United Kingdom. Only cancer cells sensitive to the anti-proliferative effects of 5F 203 deplete this drug candidate from nutrient media. 5F 203 induces cell cycle arrest, cytochrome P450 1A1 (CYP 1A1) mRNA and protein expression, and is metabolized into reactive electrophilic species that can covalently bind to DNA and form adducts in sensitive (i.e., MCF-7) but not in resistant (i.e., MDA-MB-435) breast cancer cells. METHODS: In this present study, we investigated additional anticancer effects of 5F 203 in MCF-7 cells. In addition, we sought to determine if cells deficient in the xeroderma pigmentosum D gene, a gene critical in DNA repair, would show greater sensitivity to the cytotoxic effects of 5F 203 than those complemented with XPD. RESULTS: Alkaline Elution revealed that 5F 203 induced single-strand breaks and DNA-protein cross-links in sensitive MCF-7 cells. In contrast, we detected no double-strand breaks or protein-associated strand breaks typically associated with topoisomerase I (top1) or topoisomerase II (top2) inhibition. In addition, 5F 203 was unable to trap top1- or top2-DNA cleavage complexes in MCF-7 cells. 5F 203 induced cell cycle arrest in MCF-7 cells following DNA damage after brief exposures. Cells deficient in the nucleotide excision repair xeroderma pigmentosum group D (XPD) gene displayed sensitivity to 5F 203 while cells complemented with XPD displayed resistance to 5F 203. CONCLUSION: These data suggest that the anti-cancer activity of 5F 203 depends upon targets other than top1 or top2 and on the ability of this benzothiazole to form single-strand breaks and DNA-protein cross-links in cancer cells.

Synthesis, structure-activity relationship, and p210(bcr-abl) protein tyrosine kinase activity of novel AG 957 analogs.

A series of novel, sterically hindered lipophilic analogs of AG 957 was designed and synthesized as potential protein tyrosine kinase (PTK) inhibitors. The in vitro activity, in vivo anti-leukemia activity, and pharmacology of these PTK inhibitors were studied. Some aspects of the structure-activity relationship associated with the carboxylic acid, phenol ring, and linker modifications are discussed. We have demonstrated that the 1,4-hydroquinone moiety is essential for activity and that sterically hindered esters contribute to enhanced in vivo efficacy. Adaphostin (NSC 680410) has emerged as the improved compound with the maximum in vivo anti-leukemia hollow fiber activity, concordant with the original lead compound AG 957. Currently, adaphostin is undergoing preclinical toxicology studies.

The antitumor drug candidate 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole induces NF-kappaB activity in drug-sensitive MCF-7 cells.

2-(4-Amino-3-methylphenyl)-5-fluoro-benzothiazole (5F 203) potently inhibits MCF-7 breast cancer cell growth in part by activating the aryl hydrocarbon receptor (AhR) signaling pathway. Ligands for the AhR (i.e. dioxin) have also been shown to modulate the NF-kappaB signaling cascade, affecting physiological processes such as cellular immunity, inflammation, proliferation and survival. The objective of this study was to investigate the effect of 5F 203 treatment on the NF-kappaB signaling pathway in breast cancer cells. Exposure of MCF-7 cells to 5F 203 increased protein-DNA complex formation on the NF-kappaB-responsive element as determined by electrophoretic mobility shift assay, but this effect was eliminated in MDA-MB-435 cells, which are resistant to the antiproliferative effects of 5F 203. An increase in NF-kappaB-dependent transcriptional activity was confirmed by a significant increase in NF-kappaB-dependent reporter activity in sensitive MCF-7 cells, which was absent in resistant MDA-MB-435 cells and AhR-deficient subclones of MCF-7 cells. Inhibition of NF-kappaB activation enhanced the increase in xenobiotic response element-dependent reporter activity in MCF-7 cells when treated with 5F 203. The drug candidate 5F 203 also induced mRNA levels of IL-6, an NF-kappaB-responsive gene, in MCF-7 cells, but not in MDA-MB-435 cells, as determined by quantitative RT-PCR. These findings suggest that 5F 203 activation of the NF-kappaB signaling cascade may contribute to 5F 203-mediated anticancer activity in human breast cancer MCF-7 cells.

Poly(ethylene glycol) prodrugs of the CDK inhibitor, alsterpaullone (NSC 705701): synthesis and pharmacokinetic studies.

Two methods were devised to conjugate PEG to alsterpaullone (NSC 705701) via the N of the indole ring portion of the molecule. In the first approach, activation of the indole was accomplished by reaction with p-nitrophenyl chloroformate to produce a reactive carbamate that was then condensed with a mono blocked diamine to form a urea bond followed by deblocking and conjugation to PEG. The second route used the anion of the indole and produced a carbamate bond. Both compounds were highly water soluble, were stable in buffer, and released alsterpaullone in vitro and in vivo. Studies were conducted in mice to investigate the influence of PEGylation on the plasma pharmacokinetics of alsterpaullone. The total plasma clearance rate was decreased up to 32-fold, and the biological halflife lengthened up to 8-fold when alsterpaullone was injected i.v. as a PEG-conjugate and compared to injection of the unconjugated compound. The most pronounced effect on the pharmacokinetics of alsterpaullone was produced by a 40-kDa PEG urea-linked conjugate. When the 40- and 20-kDa urea-linked conjugates were administered by i.p. injection, high relative bioavailability (46% and 99%, respectively) of alsterpaullone was observed.

Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles induce CYP1A1 expression, become metabolized, and bind to macromolecules in sensitive human cancer cells.

Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles possess potent antiproliferative activity against certain cancer cells, similar to the unfluorinated 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495). In "sensitive" cancer cells, DF 203 is metabolized by, can induce expression of, and binds covalently to CYP1A1. Metabolism appears to be essential for its antiproliferative activity through DNA adduct formation. However, a biphasic dose-response relationship compromises its straightforward development as a chemotherapeutic agent. We investigated whether fluorinated benzothiazoles inhibit cancer cell growth without the biphasic dose-response, and whether the fluorinated benzothiazoles are also metabolized into reactive species, with binding to macromolecules in sensitive cancer cells. One fluorinated benzothiazole, 2-(4-amino-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) did exhibit potent, antiproliferative activity without a biphasic dose-response. The fluorinated benzothiazoles were also metabolized only in cells, which subsequently showed evidence of cell death. We used microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochromes P450 (CYP1A1, CYP1A2, or CYP1B1) to clarify the basis for fluorinated benzothiazole metabolism. 5F 203 induced CYP1A1 and CYP1B1 mRNA expression in sensitive breast and renal cancer cells, whereas 5F 203 induced CYP1A1 mRNA but not CYP1B1 mRNA expression in sensitive ovarian cancer cells. 5F 203 did not induce CYP1A1 or CYP1B1 mRNA expression in any "resistant" cancer cells. The fluorinated benzothiazoles induced CYP1A1 protein expression exclusively in sensitive cells. [14C]5F 203 bound substantially to subcellular fractions in sensitive cells but only minimally in resistant cells. These data are concordant with the antiproliferative activity of fluorinated benzothiazoles deriving from their ability to become metabolized and bind to macromolecules within sensitive cells.

17-(Allylamino)-17-demethoxygeldanamycin activity in human melanoma models.

17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) is a semisynthetic antitumor agent, which has entered phase I/II clinical trials. Melanoma cell lines in the NCI in vitro screen (mean GI50 = 84 nM) were relatively sensitive to the agent, which was therefore tested in vivo in four s.c. growing human melanoma xenografts (MEXF 276, 989, 462 and 514) in athymic mice. 17-AAG markedly inhibited tumor growth at doses of 80 (maximum tolerated dose) and 60 mg/kg/day in a qd x 5 (h: 0, 6; i.p.) schedule in two of four xenograft models. Cell lines derived from the 17-AAG-sensitive MEXF 276 and -resistant MEXF 514 melanomas, MEXF 276L and 514L, were chosen to study the effects of 17-AAG on its target Hsp90 as well as the Hsp90 'client' protein c-Raf-1 in vitro. Cells were exposed to drug concentrations which just cause total growth inhibition (total growth inhibition = 375 nM in MEXF 276L and 10 microM in MEXF 514L). Pharmacokinetic determinations confirmed that 17-AAG concentrations producing growth inhibition invitro are readily achievable in vivo at the MTD (AUC0- infinity 1068 microM x min). Whilst 17-AAG treatment did not affect Hsp90 expression in the relatively resistant MEXF 514L cells, it caused a rapid transient decline in the markedly sensitive MEXF 276L cell line. In contrast, Hsp72 expression increased. Following Hsp90 depletion at 2-8 h in MEXF 276L cells, down-regulation of c-Raf-1 was seen starting at 16 h after drug addition. In MEXF 276 xenograft tissues treated with effective dose levels, loss of Hsp90 was seen and was associated with occurrence of apoptotic figures. The apoptotic index rose from 9% after 48 h, greater than 12% at 72 h to 45% at 10 days. These data support the hypothesis that in some melanoma models, a very good response (e.g. with tumor regressions) to 17-AAG may be associated with modulation of Hsp90 expression. The expression of this target should be followed in clinical studies with 17-AAG.

Clinical pharmacology of flavopiridol following a 72-hour continuous infusion.

BACKGROUND: Flavopiridol, a novel flavone derivative, inhibits cyclin-dependent kinase-1. We initiated a Phase I trial in patients with refractory solid tumors to determine the maximum tolerated dose and characterize the adverse effect profile. OBJECTIVE: To characterize the clinical pharmacology of flavopiridol. METHODS: Serial plasma samples were collected and analyzed by HPLC using electrochemical detection. The pharmacokinetics were analyzed by noncompartmental analysis. Enterohepatic recirculation was studied by analyzing fecal samples, with an attempt to correlate cholecystokinin and post-infusional peak concentrations. The plasma protein binding was studied using equilibrium dialysis. RESULTS: Seventy-six patients were treated with flavopiridol at 13 dose levels for a total of 504 cycles of treatment. The average steady-state concentration was 26.5 and 253 nM at 4 and 122.5 mg/m2, respectively. The clearance ranged from 49.9 to 2943 mL/min, with nonlinearity at doses >50 mg/m2/d. A post-infusional increase in plasma flavopiridol concentrations was noted in a subset of patients and generally occurred between 3 and 24 hours after the end of infusion. Flavopiridol was found in fecal matter, suggesting enterohepatic recirculation. There was nonsaturable plasma protein binding of flavopiridol (fu = 6%). CONCLUSIONS: The dose-limiting toxicity for the Phase I trial of flavopiridol was secretory diarrhea. We failed to identify a clear relationship between dose or concentration and diarrhea. At 50 and 78 mg/m2/d, the mean steady-state plasma concentrations were 278 and 390 nM. These concentrations were well above those noted for in vitro antiproliferative activity. Nonlinear elimination was observed at doses above 50 mg/m2/d, and postinfusional peaks appear to be related to enterohepatic recirculation.

Preclinical evaluation of amino acid prodrugs of novel antitumor 2-(4-amino-3-methylphenyl)benzothiazoles.

Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent antitumor properties in vitro and in vivo. Elucidation of the mechanism of action of this structurally simple class of compounds has occurred in parallel with selection of a candidate clinical agent. Antitumor benzothiazoles induce and are biotransformed by cytochrome P 450 1A1 to putative active, as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. Amino acid conjugation to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles has been used to overcome limitations posed by drug lipophilicity. Water soluble, chemically stable prodrugs rapidly and quantitatively revert to their parent amine in mice, rats, and dogs in vivo. Plasma concentrations of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (2) regenerated from the lysylamide prodrug (2b), sufficient to elicit cytocidal activity against ZR-75-1 and T47D human mammary carcinoma cell lines persist > 6 h. The growth of breast (MCF-7) and ovarian (IGROV-1) xenograft tumors is significantly retarded by 2b. Manageable toxic side effects are reported from preclinically efficacious doses of 2b. Cytochrome P 450 1A1 protein expression, selectively induced in sensitive carcinoma cells, was detected in MCF-7 and IGROV-1 tumors 24 h after treatment of mice with 2b (20 mg/kg). The lysyl amide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is potentially suitable for clinical evaluation.

Identification of the major metabolite of 2,5-bis(5-hydroxymethyl-2-thienyl)furan (NSC 652287), an antitumor agent, in the S9 subcellular fraction of dog liver cells.

Alpha-Terthienyl (1) is a trithiophene found widely distributed in plants. Other naturally occurring trithiophenes are less widely distributed, but nonetheless exhibit potent antiviral and cytotoxic activities. A synthetic analog of 1, 2,5-bis(5-hydroxymethyl-2-thienyl)furan (2; NSC 652287) has recently been shown to possess exceptional activity and selectivity against several cell lines of the National Cancer Institute (NCI) anticancer drug screen. When incubated with the S9 subcellular fraction of dog liver cells, the concentration of 2 was observed to decline as a function of time, with a concomitant increase in a significant, time-dependent concentration of an unknown entity. The results of electron-ionization mass spectrometric analysis of the metabolite indicate an increase in 14 amu over that of 2, leading to suspicions that either an oxidation or a methylation had occurred. Results of differential derivatization and accurate mass analysis allow us to propose that metabolism of 2 involves the biotransformation of one of the two hydroxymethyl groups of 2 into a carboxylic acid functionality. This is further supported by separate experiments involving chemical oxidation and S9 incubation of 5-[5-[5-hydroxymethyl-2-thienyl]-2-furanyl]-2-thiophenecarboxaldehyde: comparing the mass spectra and gas chromatographic retention times of the resulting products to those of the identified metabolite of 2 show all to be the same.

Functional assessment. Easy-to-use screening tools speed initial office work-up.

The mnemonic DEEP-IN stands for a series of easy-to-administer, office-based screening tests designed to streamline initial assessment of the geriatric patient. These screens can quickly identify signs of delirium, dementia, depression, and adverse drug effects; vision and hearing deficits; risk for future impairments in activities of daily living; incontinence, and malnutrition. The author developed the mnemonic from personal experience and from an evidence-based application of validated geriatric and geropsychiatric studies.

Requirements of Cyc2p and the porin, Por1p, for ionic stability and mitochondrial integrity in Saccharomyces cerevisiae.

It was previously demonstrated that Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein; that the cyc2-Delta2 deletion lacking the entire gene causes a diminution to only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo-cytochrome c; that the deletion causes a defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources; and that this defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a nonionic compound. We report that por1-Delta strains lacking the mitochondrial porin, Por1p, but not por2-Delta strains lacking the related porin, share some phenotypes similar to the cyc2-Delta2 strain, including hypersensitivity to KCl in glycerol medium. Moreover, spontaneous swelling in the presence of ATP was detected in mitochondria from the cyc2-Delta2 strain, while swelling could be detected in mitochondria from the other strains only after the addition of KCl. Thus, highly unspecific membrane permeation may be triggered by ATP in the cyc2-Delta2 strain. We suggest that Por1p and Cyc2p, in addition to their own unique functions, serve to maintain the osmotic stability of mitochondria, but by different mechanisms.