Expanded Characterization of a Hemi-Body Shielded Göttingen Minipig Model of Radiation-induced Gastrointestinal Injury Incorporating Oral Dosing Procedures.

In collaboration with the Biomedical Advanced Research and Development Authority (BARDA), the authors recently conducted a pilot study in a hemi-body shielded model of radiation-induced gastrointestinal (GI) injury in Göttingen minipigs following exposure to radiation dose levels between 8-16 Gy. Herein, the impact of oral dosing procedures is assessed, as well as the specific causes of death in animals exposed to radiation doses of 14 and 16 Gy (n = 64; 32 male, 32 female, between 6 and 8 mo of age). Oral dosing using a 2-tablet placebo system comprised of both immediate release and enteric-coated tablets starting 24 h post-irradiation resulted in inhibited gastric emptying of the enteric-coated tablets, which were found to be retained in the stomach and/or regurgitated. This finding appears to be species-specific, as similar findings have not been reported for other large animal species (e.g., non-human primates). Mortality was primarily dictated by decreased activity, body weight loss (>35%), and/or respiratory distress, despite shielding of the lung. The cause of respiratory distress in animals that were pre-terminally euthanized varied according to the timing of death, with interstitial inflammation and extensive fibrosis observed >20 days post-irradiation. Kidney damage was also identified in most animals after day 10. Changes in the GI tract were consistent with previous studies and included collagen deposition/fibrosis. Observations of inflammatory infiltrates and interstitial inflammation/fibrosis in both shielded and unshielded organs support a strong secondary inflammatory syndrome post-irradiation.

Pilot Study of Radiation-induced Gastrointestinal Injury in a Hemi-body Shielded Göttingen Minipig Model.

Development of medical countermeasures (MCMs) for gastrointestinal (GI) injury following acute radiation exposure requires well-characterized models that can assess not only survival but also secondary endpoints, including structural and functional characteristics of GI damage and recovery that ultimately contribute to long-term survival. The authors conducted a pilot study in a hemi-body shielded Göttingen minipig model of radiation-induced GI injury that enables radiation damage to the GI tract to be evaluated and reduces the potential for hemorrhage and/or damage in other more sensitive organ systems. With shielding of the head, chest, and front legs, radiation dose levels of 14 Gy were required to see significant GI-related morbidity, while dose levels of 16 Gy resulted in significant mortality by day 45 post-irradiation. Periodic scheduled necropsies showed significant reduction in and slow recovery of intestinal crypt count at 14 and 16 Gy. Intestinal proliferative activity was initially increased and then gradually decreased over the course of the study. Histological evidence of marked inflammatory infiltrates was noted in the GI tract at day 5, while collagen deposition, indicative of fibrosis, was observed as early as day 15, peaking at day 30. The radiation dose-responsive indicators of GI damage identified in this model (i.e., intestinal crypt count and proliferative activity) may serve as useful endpoints for evaluation of the efficacy of potential MCMs.

Citrulline as a Biomarker for Gastrointestinal-Acute Radiation Syndrome: Species Differences and Experimental Condition Effects.

Animal models of hematopoietic and gastrointestinal acute radiation syndromes (ARS) have been characterized to develop medical countermeasures. Acute radiation-induced decrease of intestinal absorptive function has been correlated to a decrease in the number of intestinal crypt cells resulting from apoptosis and enterocyte mass reduction. Citrulline, a noncoded amino acid, is produced almost exclusively by the enterocytes of the small intestine. Citrullinemia has been identified as a simple, sensitive and suitable biomarker for radiation-induced injury associated with gastrointestinal ARS (GI-ARS). Here we discuss the effect of radiation on plasma citrulline levels in three different species, C57BL/6 mice, Göttingen minipigs and rhesus nonhuman primates (NHPs), measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). The effects of experimental study conditions such as feeding and anesthesia were also examined on plasma citrulline levels in the NHPs. Both the mice and Göttingen minipigs were partial-body irradiated (PBI) with doses from 13-17 Gy and 8-16 Gy, respectively, whereas NHPs were total-body irradiated (TBI) with doses from 6.72-13 Gy. Blood samples were taken at different time points and plasma citrulline levels were measured in the three species at baseline and after irradiation. Basal plasma citrulline concentrations (mean ± SEM) in mice and minipigs were 57.8 ± 2.8 µM and 63.1 ± 2.1 µM, respectively. NHPs showed a basal plasma citrulline concentration of 32.6 ± 0.7 µM, very similar to that of humans (∼40 µM). Plasma citrulline progressively decreased after irradiation, reaching nadir values between day 3.5 and 7. The onset of citrulline recovery was observed earlier at lower radiation doses, while only partial citrulline recovery was noted at higher radiation doses in minipigs and NHPs, complete recovery was noted in mice at all doses. Plasma citrulline levels in NHPs anesthetized with ketamine and acepromazine significantly decreased by 35.5% (P = 0.0017), compared to unanesthetized NHPs. In the postprandial state, citrulline concentrations in NHPs were slightly but significantly decreased by 12.2% (P = 0.0287). These results suggest that plasma citrulline is affected by experimental conditions such as anesthesia and feeding.

Biomolecular simulations: recent developments in force fields, simulations of enzyme catalysis, protein-ligand, protein-protein, and protein-nucleic acid noncovalent interactions.

Computer modeling has been developed and widely applied in studying molecules of biological interest. The force field is the cornerstone of computer simulations, and many force fields have been developed and successfully applied in these simulations. Two interesting areas are (a) studying enzyme catalytic mechanisms using a combination of quantum mechanics and molecular mechanics, and (b) studying macromolecular dynamics and interactions using molecular dynamics (MD) and free energy (FE) calculation methods. Enzyme catalysis involves forming and breaking of covalent bonds and requires the use of quantum mechanics. Noncovalent interactions appear ubiquitously in biology, but here we confine ourselves to review only noncovalent interactions between protein and protein, protein and ligand, and protein and nucleic acids.

Elucidating the nature of enzyme catalysis utilizing a new twist on an old methodology: quantum mechanical-free energy calculations on chemical reactions in enzymes and in aqueous solution.

How do enzymes achieve very large rate enhancements compared to corresponding uncatalyzed reactions in solution? We present a computational approach which combines high-level ab initio quantum mechanical calculations with classical free energy calculations to address this question. Our calculations lead to accurate estimates of DeltaG for both trypsin and catechol O-methyltransferase-catalyzed and reference uncatalyzed reactions and give new insights into the nature of enzyme catalysis. The same methodology applied to steps in the catalytic mechanism of citrate synthase further supports the conclusion that one need not invoke special concepts such as "low-barrier hydrogen bonds" or "pK(a) matching" to explain enzyme catalysis.

Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models.

A historical perspective on the application of molecular dynamics (MD) to biological macromolecules is presented. Recent developments combining state-of-the-art force fields with continuum solvation calculations have allowed us to reach the fourth era of MD applications in which one can often derive both accurate structure and accurate relative free energies from molecular dynamics trajectories. We illustrate such applications on nucleic acid duplexes, RNA hairpins, protein folding trajectories, and protein-ligand, protein-protein, and protein-nucleic acid interactions.

Calculation and prediction of binding free energies for the matrix metalloproteinases.

The zinc-dependent matrix metalloproteinases are drug targets of interest for diseases ranging from arthritis to cancer. Unfortunately, the use of computational rational drug design has been limited by the challenges introduced by the zinc center. We present an extension of the MM/PB/SA methodology which allows us to calculate the relative binding energies of six known nanomolar carboxylate ligands of MMP-1. We are able to rank the neutral and charged ligands correctly. We further illustrate the utility of our approach by modifying the best-binding ligand of our set and predicting a better binding ligand.