Charnell I. Sommers

Fibrin sealant in corneal stem cell transplantation.

PURPOSE To determine if transplanted corneal epithelial stem cells are safely and efficiently attached to the deficient limbal niche with use of fibrin sealant. The primary outcome is measured with respect to the stability of the transplant, with secondary qualitative evaluations of inflammation, patient comfort, speed of operation, and incidence of complications. METHODS This retrospective case study examined a total of 114 corneal stem cell reconstructions performed in 95 patients from 1996 to 2004 using corneal stem cells primarily, with a minority of amnion alone, or both. Fibrin sealant was used as the only technique of stem cell adhesion for limbal reconstruction for primary or recurrent pterygia and various stem cell-deficient diseases from 2000 to 2004. RESULTS The fibrin sealant group showed 1 small recurrence of pterygium but no complications. With sutures, there were 3 recurrences in the pterygia group. After completion of all surgical procedures, all patients were free of pterygia. Miscellaneous stem cell deficiencies were included to demonstrate that corneal stem cell transplants can be used in other corneal procedures in addition to pterygia. CONCLUSIONS Fibrin sealant alone effectively and safely attached corneal stem cell transplants to the limbal niche. The additional qualitative observations of a reduction in operation time, postoperative pain, and inflammation augurs for more extensive use of fibrin sealants in ophthalmology.

Preclinical Pharmacology of BA-TPQ, a Novel Synthetic Iminoquinone Anticancer Agent

Marine natural products and their synthetic derivatives represent a major source of novel candidate anti-cancer compounds. We have recently tested the anti-cancer activity of more than forty novel compounds based on an iminoquinone makaluvamine scaffold, and have found that many of the compounds exert potent cytotoxic activity against human cancer cell lines. One of the most potent compounds, BA-TPQ [(11,12),7-(benzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one], was active against a variety of human cancer cell lines, and inhibited the growth of breast and prostate xenograft tumors in mice. However, there was some toxicity noted in the mice following administration of the compound. In order to further the development of BA-TPQ, and in a search for potential sites of accumulation that might underlie the observed toxicity of the compound, we accomplished preclinical pharmacological studies of the compound. We herein report the in vitro and in vivo pharmacological properties of BA-TPQ, including its stability in plasma, plasma protein binding, metabolism by S9 enzymes, and plasma and tissue distribution. We believe these studies will be useful for further investigations, and may be useful for other investigators examining the use of similar compounds for cancer therapy.

Synthetic complementary peptides inhibit a neutrophil chemoattractant found in the alkali-injured cornea.

Purpose. We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense–antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. Methods. The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. Results. The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 &mgr;M). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 &mgr;M) but had no effect on LTB4. Conclusions. A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

NMR conformational analysis of cis and trans proline isomers in the neutrophil chemoattractant, N-acetyl-proline-glycine-proline.

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.

Characterization of chemical gradients in the collagen gel-visual chemotactic assay

Chemical gradients developing in a collagen gel-visual chemotactic assay (CG-VCA) for PMNs were evaluated by theoretical and experimental methods. First, a video image analysis system was used to establish the diffusion coefficients of bromophenol blue (BPB) through the membrane (D1) and across the collagen gel (D2) in a capillary tube apparatus used only for this purpose. The diffusion coefficients of BPB and the geometry of the CG-VCA system were then used to develop a mathematical model, estimating theoretical gradients in the CG-VCA system. In addition, gradients in the CG-VCA system were characterized experimentally by employing BPB as the source and determining the concentration profiles of BPB in the chemotactic compartment by video image analysis. A relative error of approximately 21% exists between the theoretical and experimental gradients for both 0.5 and 1.0 mM source concentrations of BPB. This favorable comparison demonstrates reliability in predicting chemical gradients for the CG-VCA system. The mathematical model was then used to predict gradients using nanomolar and micromolar concentrations of low molecular weight chemoattractants. Analysis of these specific gradients showed that gradients were steep enough to be detected by PMNs in the collagen gel during the observation period used in previous experiments. The determination of BPB gradients in the CG-VCA system illustrates the utility of this system.

Identification of Protein-Protein Interaction Inhibitors Targeting Vaccinia Virus Processivity Factor for Development of Antiviral Agents

ABSTRACT Poxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.

A visual assay for quantitating neutrophil chemotaxis in a collagen gel matrix

The chemotactic behavior of polymorphonuclear leukocytes (PMNs) suspended in a three-dimensional gel of native collagen fibers was analyzed using a new visual assay aided by computer assisted tracking. Cell behavior was observed in a 7 microliters chamber closed at either end with capillary tubes tipped with dialysis membrane. The chemoattractant, LTB4, was placed in one capillary tube and the control substance in the opposite tube. Under microscopic observation neutrophils were videotaped, their images digitized, and the x and y coordinates of the cell centroids captured at 30 s intervals for 15 min and subsequently analyzed. The data generate a global perspective of neutrophil behavior in a medium simulating a collagenous tissue. The results show that when leukotriene B4 was substituted for HBSS the PMN population underwent chemotactic displacement. PMN chemotaxis was increased greatly when the concentration of LTB4 was increased from 10 nM to 1 microM in separate experiments. This result was partly achieved by movement of an increasing percentage of the PMN population, less frequent stops, and longer durations of motility for individual cells. The most dramatic effect of LTB4 on neutrophil behavior was a large increase in directional movement toward the chemotactic source. The effects of LTB4 fell dramatically when the gradient source concentration was increased to 10 microM. The visual assay described here provides clear evidence that LTB4 induces true neutrophil chemotaxis in a collagenous matrix.

Development and validation of an HPLC method for quantitation of BA-TPQ, a novel iminoquinone anticancer agent, and an initial pharmacokinetic study in mice.

We herein describe the development and validation of an HPLC method for the quantitation of 7-(benzylamino)-1,3,4,8-tetrahydropyrrolo [4,3,2-de]quinolin-8(1H)-one (BA-TPQ), a newly synthesized iminoquinone anticancer agent. BA-TPQ was extracted from plasma and tissue samples by first precipitating proteins with acetonitrile followed by a liquid-liquid extraction with ethyl acetate. Chromatographic separation was carried out using a gradient flow rate on a Zorbax SB C(18) column, and the effluent was monitored by UV detection at 346 nm. The method was found to be precise, accurate, and specific, with a linear range of 3.91-1955.0  ng/mL in plasma, 19.55-1955.0  ng/mL in spleen, brain, and liver homogenates and 19.55-3910.0  ng/mL in heart, lung and kidney homogenates. The method was stable under all relevant conditions. Using this method, we also carried out an initial study determining plasma pharmacokinetics and tissue distribution of BA-TPQ in mice following intravenous administration. In summary, this simple and sensitive HPLC method can be used in future preclinical and clinical studies of BA-TPQ.

L-arginine-threonine-arginine (RTR) tetramer peptide inhibits ulceration in the alkali-injured rabbit cornea.

Purpose: Proline-glycine-proline (PGP) peptides have been identified as inflammatory mediators initiating neutrophil invasion into alkali-injured cornea. The complementary peptide, arginine-threonine-arginine (RTR), has been shown to bind to the PGP sequence and impede neutrophil infiltration. A prior study showed that L-RTR tetramer and D-RTR tetramer, used alternately (14 times a day), resulted in significantly reduced incidences of corneal ulceration and severity. The purpose of this experiment is to determine the effectiveness of both tetramers, used separately, compared with control. Methods: Rabbit corneas were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of 3 groups: 1) phosphate-buffered saline (PBS), 2) 1.5 mM L-RTR, or 3) 800 μM D-RTR. One drop of each was administered hourly (14 times a day) for 36 days. Additional studies were done to assess neutrophil infiltration into corneas with and without RTR treatment. Results: The severity of corneal ulceration in both RTR groups was statistically significantly different from the 21st day of the experiment to the end. As a result of ulcers healing in the L-RTR group, there was a statistically significant reduction in the number of ulcers beginning on day 22 versus control. Although there was healing in the D-RTR group, the incidence of ulcers was not significantly different from control or L-RTR. Morphometric analysis revealed decreased neutrophil (PMN) invasion with RTR treatment compared with PBS control. Conclusions: Binding of the PGP molecules by RTR tetramer seems to deprive the cornea of this neutrophilic chemotactic stimulus, leading to a reduction in the severity and incidence of corneal ulceration.

Abstract 3605: Enterohepatic circulation of indolecarboxamide ML-970 (NSC 716970), an investigative anticancer agent

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC ML-970 (NSC 716970), an analog of CC-1065 (a DNA minor-groove binding agent) is under preclinical investigation as a novel anticancer agent. The compound has shown in vitro and in vivo anti-cancer activity. As part of the preclinical evaluation of this agent, we carried out pharmacokinetic studies of ML-970 in CD-1 and nude mice. Of interest, we observed that there was minimal urinary and fecal excretion of the agent within the first 24 hr and relatively low concentrations of the compound in tissues examined (lungs, kidneys, heart, spleen, brain). Additionally, the compound was still detectable at low levels in the plasma for at least 8 hr, and in tissues for at least 24 hr after administration, regardless of the routes of administration. Although high levels of plasma protein binding of the compound may be partially responsible, it was apparent that other factors might be contributing. To examine the underlying mechanism, we performed a pharmacokinetic study evaluating the distribution of the compound in tissues, organs, and organ contents (plasma, liver, lungs, kidneys, spleen, heart, brain, stomach, stomach contents, small intestine, small intestine contents, large intestine, large intestine contents, gallbladder, muscle, urine and feces) after i.v. and oral administration. We found the highest levels of the compound in the gallbladder, small intestine, small intestine contents, large intestine contents and liver, regardless of route of administration. Concentrations of 314.7μg/g, 1383.5μg/g, and 392.6μg/g for 0.5, 2, and 8 hr after i.v. injection, and 28.0μg/g, 313.1μg/g, and 123.7μg/g for 0.5, 2, and 8 hr after oral administration, respectively, were observed for the gallbladder. Average concentrations higher than 19 μg/g were found in the other tissues for at least one time point (range: 19.3μg/g to 286.2μg/g). The high concentrations present in gastrointestinal and hepato-biliary organs, persistence of the compound, and relatively low levels of urinary and fecal excretion during the first 24 hr, indicate that the compound undergoes enterohepatic circulation. These results should be useful for interpretation of future preclinical and clinical developmental studies of ML-970. (This work was supported by NCI Contract N01-CM-52207.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3605.