**5.6 Clinical trials**

highest dosage (40mg kg<sup>1</sup>

*Weight Management*

driven by ROS clearance.

**5.4 Other in vivo studies with animal models**

mg kg<sup>1</sup>

attenuated.

**280**

ical doses of GSPE (375 mg kg<sup>1</sup>

anti-inflammatory cytokine IL-13.

**5.5 Human ex vivo studies**

), were comparable to those of mesalazine (200

) in a rat model of mild intestinal dysfunction

) and pharmacolog-

), a COX inhibitor. The authors suggest that these effects were largely due

The IL-10 deficient mouse is a classic knockout model that develops spontaneous colitis under pathogen-free conditions. Some authors have explored the influence of GSPE in this model, supplementing colitic animals with 0.1–1 g 100 g<sup>1</sup> of dry feed weight for 12–16 days [86, 87]. These studies evidenced a reduction of multiple inflammatory markers in the jejunum and colon, such as TNF-α, IL-1β, IL-6, and IFN-γ gene expressions, as well as MPO activity. This anti-inflammatory effect was associated with the inhibition of the NF-κB signaling. Interestingly, GSPE supplementation also increased the density of goblet cells in the jejunum of treated animals, suggesting that there is an alternative mechanism by which inflammation is

to the reduction in activated macrophages infiltrating colonic tissues, probably

Cardoso et al. [13] recently tested both dietary (75 mg kg<sup>1</sup>

induced by intraperitoneal injection of LPS. GSPE was administered daily by oral gavage for 15 days prior to LPS-induced intestinal dysfunction. LPS enhanced intestinal permeability and induced both oxidative stress and inflammation. GSPEtreated animals reduced OVA permeation to the circulation, MPO activity and COX-2 in the small intestine tissues, and reactive oxygen species (ROS) levels in the colon. Furthermore, a gene expression analysis with a low-density microarray technique revealed that unlike the dietary dose of GSPE, the pharmacological dose had a striking effect on the LPS-gene expression profile, showing a strong modulation of multiple genes associated with chemokines and ILs, including upregulation of the

Although the use of animal models is the predominate approximation at preclinical stages for testing novel therapies in intestinal permeability, there is a strong trend in the industry towards replacing animal experiments with human cellculture based models [92, 93]. Nevertheless, advantages related to the usefulness of in vitro models for screening of bioactives and exploring action mechanisms, are offset by limitations regarding the mimicking of the in vivo situation and translation to the human [103]. Thus, some human ex vivo models have been proposed to test immunomodulatory properties of drug candidates in intestinal explants from IBD patients [104, 105]. Intestinal function can also be studied with UCh-based protocols. The UCh system consists of two halves with an opening between them, where mucosal tissue is adapted, thus isolating the apical and basolateral sides of the tissue. This technique has been applied for studying drug absorption [106] and secretion of enterohormones [107] in human endoscopic biopsies. An advantage of UCh models over explant-based models is that UCh models make it possible to measure the electrophysiological parameters, including TEER [106]. All these setups permit analyzing the cytokine profiling of intestinal explants or biopsies retaining their in situ conditioning in a polarized fashion [105, 108]. We have employed the UCh to determine TEER and cytokine release (TNF-α) in intestinal tissues from cafeteria diet-induced obese rats treated with GSPE [8, 15]. It could also be useful for testing the effect of bioactives on dysfunctional human intestine.

Translation of doses of PAC-rich extracts used in rodent models of intestinal dysfunction to human equivalent doses (HED) indicates that pharmacological doses (up to 5 g d<sup>1</sup> for a 60 kg person) could be required to achieve beneficial effects in clinical trials [14, 15]. Thus, the first uncertainty involved in assessing the use of PACs as therapy agents in humans, is safety. Grape seed and skin proanthocyanidin-rich extracts have been subjected to toxicological tests in rats to determine their safety for use in functional foods [109–111]. In these studies, the median lethal dose (LD50) was found to be greater than 5000 mg kg<sup>1</sup> bw (HED of ≈50 g) when administered once by oral gavage, and 1400–2000 mg kg<sup>1</sup> d<sup>1</sup> (HED of ≈14–20 g d<sup>1</sup> ) was found to be the no-observed-adverse-effect level (NOAEL) for systemic toxicity in sub-chronic administration. A recent study evaluated the safety and tolerability of GSPE intake (up to 2.5 g d<sup>1</sup> ) in a small number of healthy adults for a 4-week period and found a good tolerability without adverse effects on hematological or biochemical parameters [47].

To date, there are few clinical studies that evaluate the influence of PACs on intestinal inflammatory conditions. A clinical study revealed that the postprandial increase of plasma LPS associated with the intake of a high-fat meal was significantly reduced in obese subjects who consumed 1 g of GSPE [112]. As translocation of LPS to the circulation is considered an indicator of intestinal permeability and a critical factor in the appearance of systemic low-grade inflammation in patients with metabolic syndrome [113], reduction of postprandial endotoxemia could be particularly interesting from a therapeutic perspective. Large double-blind clinical studies need to be conducted to provide more information on PAC clinical efficacy in intestinal dysfunction so that these phytochemicals can be used therapeutically to improve intestinal health in obese and IBD individuals.
