**4. THDP-17: Glutaminase inhibitor in CACO-2 cultures**

It has been described as a glutaminase inhibitors Mersalyl, *N*-ethylmaleimide and 6-diazo-5 oxo-L-norleucine (DON). DON has been used in the inhibition of glutaminase in cell cultures of astrocytes in studies demonstrating the importance of GA activity in cell damage induced by ammonia. Also, in rats subjected to portocaval shunt, neomycin inhibits intestinal glutaminase activity (Hawkins, Jessy et al. 1994), although the mechanisms by which neomycin may inhibit glutaminase activity are not described. GA activity is increased in patients with high levels of nitric oxide, glucagon, or tumor necrosis factor, and in a wide number of cancer types as in liver, breast and leukemia as glutamine is implied as a factor in in cell proliferation (Perez-Gomez C 2005; Gao P 2009).

So based on the above and in the previous studies of assosiation of the presence of longlong microsatellite in the glutaminase gene and the mechanism of action of OP in gut

In this novel approach to target the altered interorgan ammonia metabolism in liver failure, OP utilizes the activity of GS to trap ammonia as glutamine, then phenylacetate facilitates its excretion as phenylacetylglutamine (Davies, Wright et al. 2009; Ytrebo, Kristiansen et al. 2009). The effectiveness of this approach with OP has been confirmed in animal models of cirrhosis and ALF. The reduction (≈50%) of plasma ammonia was associated with (a) an improvement in grade of HE in cirrhotic patients and (b) a reduction in ICP in acute liver failure. OP treatment significantly reduced ammonia concentrations, which was associated with a reduction in brain water and an increase in myoinositol levels, indicating an improvement in brain metabolism (Jalan, Wright et al. 2007; Davies 2009; Ytrebo LM 2009). In a devascularised pig model of ALF, the rise in arterial ammonia was attenuated with OP which was accompanied by a significant decrease in extracellular brain ammonia and

In our studies we included twenty-five male Sprague-Dawley rats: 4 sham operated, and 21 BDL. 5 BDL's received OP (5 days, IP 0.6 g/kg), 5 BDL's received ornithine (5 days, IP 0.6 g/kg), 5 BDL's received phenylacetate (5 days, IP 0.6 g/kg) and 6 received saline (IP). We measured plasma levels for: ammonia and standard biochemical markers. Expressions of GS, GA and ornithine amino transferase (OAT) were determined by Western-blot (expressed as a % of sham values) and enzyme activity was determined by end-point methods in liver, kidney, gut, muscle and lung. We found that plasma ammonia was decreased in BDL-OP rats vs. BDL-saline (58.97±6.02 vs. 106.2±20.56 µmol/L;P<0.05). BDL-OP rats showed increased GS expression in liver (66% BDL-OP vs. 55% BDL-saline; P < 0.01) and showed further increased levels in the muscle (153% BDL-OP vs.142% BDL-saline). OP ameliorates the BDL related increases in glutaminase expression (124%vs.163%; P<0.05) and activity (0.45±0.16mIU/mg protein BDL-OP vs. 1.14±0.046mIU/mg protein BDL-saline; P<0.01) in gut. We demonstrated that this prevention is due to effect of ornithine in glutaminase activity (0.46±0.17mIU/mg protein BDL-O vs. BDL-saline; P<0.05) and not to phenylacetate. OP treatment in BDL rats increased the conversion of glutamate to glutamine by stimulation of GS in the muscle and also resulted in normalization of glutaminase expression and activity in the gut, indicating that OP effectively restricts the production of *in vivo* ammonia in a cirrhotic. Mechanism of action of OP on the metabolism of ammonia is

It has been described as a glutaminase inhibitors Mersalyl, *N*-ethylmaleimide and 6-diazo-5 oxo-L-norleucine (DON). DON has been used in the inhibition of glutaminase in cell cultures of astrocytes in studies demonstrating the importance of GA activity in cell damage induced by ammonia. Also, in rats subjected to portocaval shunt, neomycin inhibits intestinal glutaminase activity (Hawkins, Jessy et al. 1994), although the mechanisms by which neomycin may inhibit glutaminase activity are not described. GA activity is increased in patients with high levels of nitric oxide, glucagon, or tumor necrosis factor, and in a wide number of cancer types as in liver, breast and leukemia as glutamine is implied as a factor in

So based on the above and in the previous studies of assosiation of the presence of longlong microsatellite in the glutaminase gene and the mechanism of action of OP in gut

prevention of intracranial hypertension (Ytrebo, Sen et al. 2006).

**4. THDP-17: Glutaminase inhibitor in CACO-2 cultures** 

in cell proliferation (Perez-Gomez C 2005; Gao P 2009).

shown in the Figure 2.

Fig. 2. Mechanism of action of OP in glutamine synthetase (GS) and glutaminase (GA) enzymes. GS is stimulated in muscle by glutamate increased levels. At the same time PAGN is formed and excreted in the urine. In adition, GA is restored to normal levels in the gut

glutaminase, the inhibitition of glutaminase activity is a therapeutic target in the management of hepatic encephalopathy. It is very important to investigate new molecules for the purpose of decreasing the intestinal production of ammonia, since it determines a high risk of encephalopathy and decreased survival. However, when developing these inhibitory molecules is necessary to note that complete inhibition of the enzymatic activity of glutaminase could severely damage the normal function of the enterocytes, so that molecules inhibiting the activity of glutaminase should cause a reduction in activity ie, a partial inhibition of enzyme activity, without significantly affecting the functionality of the enterocyte.

Colon carcinoma cell cultures (CACO2) have been used to test this compound. 50,000 cells per well were used in a 12-well plate with 1.2ml DMEM medium supplemented with 2mM L-Glutamine, 15% FBS, 1X antibiotic/antifungal solution, 1X non-essential amino acids (PAA Laboratories GmbH, Linz, Germany). Plates were incubated at 37°C and 5% CO2 for 24 and 48h. THDP-17 and 6-diazo-5-oxo-norleucina (DON) (Sigma, St. Louis, EE.UU.) were assayed in duplicates at 0, 5, 20 and 100µM. The glutaminase activity was assayed using the protocol described by Heini (Heini Hans G. 1987).

Using both THDP-17 and DON (positive control) 100µM, glutaminase activity was inhibited after 48h. The product THDP-17 reduces 42% of the initial glutaminase activity, while DON reduces 46% of the initial activity. Therefore THDP-17 is considered to have potential to act as a therapeutic option for the hepatic encephalopathy as a glutaminase partial inhibitor that is able to cross the cellular and mitochondrial membranes. Further studies to evaluate toxicity and *in vivo* experiments to as certain efficacy need to be done in the future. In Figure 3 it can be seen that glutaminase activity is inhibited at 48 hours with differing concentrations of THDP-17 and DON (0,5,20 and 100µM).

Fig. 3. Inhibition of GA activity following 48 hrs incubation with either THDP 17 or DON in Caco-2 cells
