Here we identify a low-cost diagnostic platform using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs, which upon binding target oligonucleotides undergo fluorescence resonance energy transfer (FRET). Using a target oligonucleotide derived from the Ebola virus (EBOV), we have derivatized PMO probes with either Alexa Fluor488 (donor) or tetramethylrhodamine (acceptor). Upon EBOV target oligonulceotide binding, observed changes in FRET between PMO probe pairs permit a 25 pM lower limit of detection; there is no off-target binding within a complex mixture of nucleic acids and other biomolecules present in human saliva. Equivalent levels of FRET occur using PMO probe pairs for single or double stranded oligonucleotide targets. High-affinity binding is retained under low-ionic strength conditions that disrupt oligonucleotide secondary structures (e.g., stem-loop structures), ensuring reliable target detection. Under these low-ionic strength conditions, rates of PMO probe binding to target oligonucleotides are increased 3-fold relative to conventional high-ionic strength conditions used for nucleic acid hybridization, with half-maximal binding occurring within 10 min. Our results indicate an ability to use PMO probe pairs to detect clinically relevant levels of EBOV and other oligonucleotide targets in complex biological samples without the need for nucleic acid amplification, and open the possibility of population screening that includes assays for the genomic integration of DNA based copies of viral RNA.

A cell membrane spanning peptide was used to increase the concentration of the IKVAV motif within damaged mouse spinal cord tissue. This peptide was injected directly to the lesion 24 hours after spinal cord compression injury. Because the membrane-spanning portion of the peptide adheres to tissue upon injection with a long half-life we hypothesized that the bioactive IKVAV sequence will provide a sustained regenerative signal at the sight of injury. Five different groups of mice were used and cellular morphology observations were undertaken using light and electron microscopy. Three surgical control groups: IKVAV, peptide and mannitol; one surgical treatment group: IKVAV-peptide; and one non-surgical control group: normal, were used in this experiment. In this study, treatment with IKVAV-peptide after SCI resulted in an increased number of protoplasmic astrocytes, large active motor neurons, and regeneration of muscle bundles followed by behavioral improvement. In this paper, we describe the cellular differences between all groups.

Spinal cord regeneration following treatment with a novel membrane-spanning peptide (MSP) expressing the isoleucine-lysine-valine-alanine-valine (IKVAV) epitope was assessed in Balb-c mice. After hemilaminectomy and compression injury, mice were treated with IKVAV, IKVAV-MSP, peptide or vehicle control. Functional improvement was assessed using modified Basso, Beattie, and Bresnahan Scale (mBBB) and spinal cord segments were studied histologically 28 days after injury. IKVAV-MSP group scores increased significantly compared with control groups after 4 weeks of observation (p < 0.05). The number of protoplasmic astrocytes, neurons and muscle bundle size in the IKVAV-MSP mice were significantly increased (p < 0.001; p < 0.05 and p < 0.007; respectively). This study demonstrates that it is possible to promote functional recovery after SCI using bioactive IKVAV presenting cell membrane-spanning peptides.

Background Osteosarcoma (OS) affects over 8000 dogs/year in the United States. The disease usually arises in the appendicular skeleton and metastasizes to the lung. Dogs with localized appendicular disease benefit from limb amputation and chemotherapy but most die within 6–12 months despite these treatments. Taurolidine, a derivative of taurine, has anti-tumor and anti-angiogenic effects against a variety of cancers. The following in vitro studies tested taurolidine as a candidate for adjuvant therapy for canine OS. Tests for p53 protein status and caspase activity were used to elucidate mechanisms of taurolidine-induced cell death. Results Taurolidine was cytotoxic to osteosarcoma cells and increased the toxicity of doxorubicin and carboplatin in vitro . Apoptosis was greatly induced in cells exposed to 125 μM taurolidine and less so in cells exposed to 250 μM taurolidine. Taurolidine cytotoxicity appeared caspase-dependent in one cell line; with apparent mutant p53 protein. This cell line was the most sensitive to single agent taurolidine treatment and had a taurolidine-dependent reduction in accumulated p53 protein suggesting taurolidine’s effects may depend on the functional status of p53 in canine OS. Conclusion Taurolidine’s cytotoxic effect appears dependent on cell specific factors which may be explained, in part, by the functional status of p53. Taurolidine initiates apoptosis in canine OS cells and this occurs to a greater extent at lower concentrations. Mechanisms of cell death induced by higher concentrations were not elucidated here. Taurolidine combined with doxorubicin or carboplatin can increase the toxicity of these chemotherapy drugs and warrants further investigation in dogs with osteosarcoma.

Osteosarcoma in dogs and humans share many similarities and the dog has been described as an excellent model to study this disease. The median survival in dogs has not improved in the last 25 years. Taurolidine has been shown to be cytotoxic to canine and human osteosarcoma in vitro. The goals of this study were to determine the pharmacokinetics and safety of taurolidine in healthy dogs and the safety of taurolidine in combination with doxorubicin or carboplatin in dogs with osteosarcoma.

Background Effective targeted therapies are needed in sarcomas, but the biological heterogeneity of these tumors has presented a challenge to clinical integration of small molecule inhibitors in sarcoma treatment. Here we outline a process to personalize therapy for sarcomas through a case study of a canine with spontaneous osteosarcoma. Procedure Rapid establishment of a primary tumor cell culture is described, followed by efficient functional characterization of the tumor that identified the Src inhibitor dasatinib as the most effective targeted therapy for this individual dog. Results Adjuvant dasatinib was administered for a total of 26 weeks following treatment with chemotherapy. Pharmacokinetic studies confirm that a therapeutic serum concentration was achieved at a tolerable dose of 0.75mg/kg/day. The canine patient remains without evidence of recurrent disease 24 months following initial diagnosis. Conclusions The approach described through this illustrative case study is broadly applicable and might be used for other solid tumors in canines as well as in humans. Pediatr Blood Cancer 2013;60:1313-1319. (c) 2013 Wiley Periodicals, Inc.

The breakdown of polyunsaturated fatty acids (PUFAs) under conditions of oxidative stress results in the formation of lipid peroxidation (LPO) products. These LPO products such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) can contribute to the development of cardiovascular and neurodegenerative diseases and cancer. Conjugation with glutathione, followed by further metabolism to mercapturic acid (MA) conjugates, can mitigate the effects of these LPO products in disease development by facilitating their excretion from the body. We have developed a quantitative method to simultaneously assess levels of 4-oxo-2-nonen-1-ol (ONO)-MA, HNE-MA, and 1,4-dihydroxy-2-nonene (DHN)-MA in human urine samples utilizing isotope-dilution mass spectrometry. We are also able to detect 4-hydroxy-2-nonenoic acid (HNA)-MA, 4-hydroxy-2-nonenoic acid lactone (HNAL)-MA, and 4-oxo-2-nonenoic acid (ONA)-MA with this method. The detection of ONO-MA and ONA-MA in humans is significant because it demonstrates that HNE/ONE branching occurs in the breakdown of PUFAs and suggests that ONO may contribute to the harmful effects currently associated with HNE. We were able to show significant decreases in HNE-MA, DHN-MA, and total LPO-MA in a group of seven smokers upon smoking cessation. These data demonstrate the value of HNE and ONE metabolites as in vivo markers of oxidative stress.

OBJECTIVE To determine pharmacokinetics and oral bioavailability of metformin in healthy horses. ANIMALS 4 adult horses. PROCEDURES 6 g of metformin was administered 3 times IV and PO (fed and unfed) to each horse, by use of a crossover design, with a 1-week washout period between treatments. Plasma metformin concentration was determined via high-pressure liquid chromatography. RESULTS Mean +/- SD distribution half-life of metformin following IV administration was 24.9 +/- 0.4 minutes with a volume of distribution of 0.3 +/- 0.1 L/kg. Mean area under the curve was 20.9 +/- 2.0 h.microg/mL for IV administration; PO administration resulted in area under the curves of 1.6 +/- 0.4 h.microg/mL in unfed horses and 0.8 +/- 0.2 h.microg/mL in fed horses. Bioavailability was determined to be approximately 7.1 +/- 1.5% in unfed horses and 3.9 +/- 1.0% in fed horses. The maximal concentration following PO administration in unfed horses was 0.4 +/- 0.1 microg/mL with a time at maximal concentration of 0.9 +/- 0.1 hours. In fed horses, maximal concentration was reduced to 0.3 +/- 0.04 microg/mL with a time at maximal concentration at 1.3 +/- 0.3 hours. CONCLUSIONS AND CLINICAL RELEVANCE The low bioavailability of metformin may explain the reported lack of clinical success in improving insulin sensitivity with metformin treatment in horses. Dosages and dose intervals previously used may have been insufficient to achieve plasma concentrations of drug comparable to the therapeutic range achieved in humans. Therefore, a larger and more frequently administered dose may be required to fully evaluate efficacy of metformin in horses.

Abstract Chlorophyll (Chla) and chlorophyllin (CHL) were shown previously to reduce carcinogen bioavailability, biomarker damage, and tumorigenicity in trout and rats. These findings were partially extended to humans, where CHL reduced excretion of aflatoxin B1 (AFB1)-DNA repair products in Chinese unavoidably exposed to dietary AFB1. However, neither AFB1 pharmacokinetics nor Chla effects were examined. We conducted an unblinded crossover study to establish AFB1 pharmacokinetic parameters among four human volunteers, and to explore possible effects of CHL or Chla cotreatment in three of those volunteers. For protocol 1, fasted subjects received an Institutional Review Board–approved dose of 14C-AFB1 (30 ng, 5 nCi) by capsule with 100 mL water, followed by normal eating and drinking after 2 hours. Blood and cumulative urine samples were collected over 72 hours, and 14C- AFB1 equivalents were determined by accelerator mass spectrometry. Protocols 2 and 3 were similar except capsules also contained 150 mg of purified Chla or CHL, respectively. Protocols were repeated thrice for each volunteer. The study revealed rapid human AFB1 uptake (plasma ka, 5.05 ± 1.10 h−1; Tmax, 1.0 hour) and urinary elimination (95% complete by 24 hours) kinetics. Chla and CHL treatment each significantly impeded AFB1 absorption and reduced Cmax and AUCs (plasma and urine) in one or more subjects. These initial results provide AFB1 pharmacokinetic parameters previously unavailable for humans, and suggest that Chla or CHL co-consumption may limit the bioavailability of ingested aflatoxin in humans, as they do in animal models.

500 We have previously shown that chlorophyll and its derivative chlorophyllin strongly reduce carcinogen bioavailability, biomarker damage, and tumorigenicity in trout and rats. These findings were partially extended to humans, where chlorophyllin was found to reduce urinary excretion of aflatoxin B1 (AFB1)-DNA repair products in rural Chinese unavoidably exposed to dietary AFB1 (Proc. Natl. Acad. Sci. USA 98:14601-14606). However, no AFB1 pharmacokinetic data were gathered in that study, and chlorophyll was not included. To address this, a small un-blinded crossover pilot study examined AFB1 pharmacokinetic parameters in human volunteers, and the effects of chlorophyll or chlorophyllin co-treatment on those parameters. Fasted subjects received an IRB-approved dose of 14C-AFB1 (30 ng, 5 nCi) by gelatin capsule with 100 ml water, alone or with 150 mg chlorophyllin or highly purified chlorophyll. Blood and total urine samples were collected at specified intervals over 72 hr, and 14C-AFB1 equivalents were determined by accelerator mass spectrometry (AMS). Each treatment was repeated 3 times for each volunteer. Total 72-hr urine aflatoxin equivalents among the three subjects ranged from 8.5-10.2 ng, and were reduced significantly by chlorophyll (range 3.9 - 6.7 ng; P

1665 We have previously shown that chlorophyll (Chl) and its derivative chlorophyllin (CHL) reduce carcinogen bioavailability, biomarker damage, and tumorigenicity in trout and rats. These findings have been partially extended to humans, where CHL was found to reduce urinary excretion of aflatoxin B1 (AFB1)-DNA repair products in rural Chinese unavoidably exposed to dietary AFB1. However, no AFB1 pharmacokinetic data were gathered by that study, and Chl was not included. To address this a small un-blinded crossover study is under way to establish AFB1 toxicokinetic parameters in human volunteers, and the effects of CHL or Chl co-treatment on those parameters. For protocol 1, fasted subjects received an IRB-approved dose of 14C-AFB1 (31.2 ng, 5 nCi) by gelatin capsule with 100 ml water, followed by normal eating and drinking after hr 2. Blood and total urine samples were collected at specified intervals over 72 hr, and 14C-AFB1 equivalents were determined by accelerator mass spectrometry. The protocol was repeated 3 times for each volunteer. Plasma sample analysis revealed a rapid absorption phase, reaching a Cmax of 0.567 ± 0.095 fg/ml at 1 hr (Tmax) (volunteer 1) and 0.534 ± 0.083 fg/ml at 0.92 ± 0.52 hr (volunteer 2). A two compartment model with first-order input and elimination was fitted to each plasma data set, yielding absorption rate constants for volunteers 1 and 2 of 7.22 ± 4.34 hr-1 and 12.46 ± 7.92 hr-1, mean plasma distribution half-lifes (t1/2 α) of 2.02 ± 0.35 hrs and 4.41 ± 2.79 hrs, terminal half-lifes (t1/2 β) of 68.52 ± 49.27 and 92.04 ± 31.18, and AUC(0-24) of 7.26 ± 2.27 pg hr/L and 7.40 ± 0.46 pg hr/L, resp. Urine eliminated over 0-24 hr contained 28.0 ± 2.2% and 35.0 ± 7.9% of the administered dose for volunteers 1 and 2, resp. Protocols 2 and 3 were similar except capsules also contained 150 mg of Chl purified from spinach, or CHL, resp. Though still in progress, single-trial results suggest CHL delayed AFB1 absorption and reduced Cmax and AUC., whereas Chla reduced absorption, Cmax and AUC. These initial results are providing AFB1 toxicokinetic parameters previously unavailable for humans, and suggest that Chl and CHL may limit the bioavailability of aflatoxin in humans as in animals. (Partially supported by NIH grants ES00210, ES03850, CA90890. The AMS work was performed under the auspices of the U.S. D. O.E. by University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48.)

Solid tumors often display metabolic abnormalities that consistently produce low pH in the extracellular space of poorly perfused tissue. These acidic regions may provide a mechanism for drug targeting. Peptides have been designed in such a manner that they exist in an anionic hydrophilic form at the pH of normal tissues, but then undergo a sharp transition to a non-ionic lipophilic form at reduced pH. Peptides were labeled with fluorescein or technicium-99m (99mTc) and evaluated in vitro and in two murine models of cancer. Our studies suggest that PAP-1, an 18 amino acid pH activated peptide with a pH of transition between hydrophilic and lipophilic forms (pT) of 6.4, will deliver fluorescein and 99mTc to tumors. Activation of PAP-1 by low pH and penetration into the plasma membrane of cells and tumors were confirmed using flow cytometry, fluorescence microscopy, and gamma scintigraphy. These results support our central hypothesis that PAP-1 may enable the selective delivery of macromolecules to tumors. This technology has potential for exploiting a common property of tumors to achieve highly specific medical intervention.

: We characterized a class of modified peptides designed to activate in the extracellular space of tumors in which pH is below 7.0 in cell culture and in tumor bearing mice. These engines change shape in a low pH environment, become lipophilic, and embed into the plasma membrane. Our studies suggest that a molecular engine with a pT of 6.8-6.9 will deliver an 8-carboxyfluoroscein ligand to the surface of tumor cells in C57blk-J6 mice bearing subcutaneously transplanted Lewis lung cell-derived tumors following an iv injection of 27 nmoles (0.1 mg) peptide with FITC-labeled ligand. These observations were confirmed by flow cytometric analysis. Proof of principle for their diagnostic utility of this technology was obtained by injection of a 99mTc labeled peptide followed by gamma scintigraphy. This technology has tremendous potential for highly specific medical intervention at the molecular scale.

c-myc antisense oligonucleotide treatment ameliorates murine ARPKD.