Protection is associated with exclusion of infection from normocytes.

<p>On day 3 blood films from control (A) and FBS0701 (B) treated mice both show infection in normocytes. On day 16 <i>P. yoelii</i> infection is largely restricted to young reticulocytes with only a few in normocytes in mice treated on day −7 (C) or day −1 (D). Quantification of blood films (E) indicated that on day 3 infected normocytes (black bars) out numbered by more than 20 fold infected reticulocytes (hatched bar). On day 16 most of the erythrocytes are either infected (hatched bars) or uninfected reticuloctyes (clear bars). By day 23 more than 80 to 90% of the erythrocytes are uninfected normocytes. Parasitemia on day 3 is reduced compared to control animals which are absent in subsequent days due to control animal death. Quantification was performed on counting at least 500 erythrocytes per mouse and averaged in the groups of 4 or number of remaining mice. Counts are represented as number of infected or uninfected normocytes or reticulocytes out of 100. Error is standard deviation of means between up to 4 mice.</p

FBS0701 interferes with artemisinin but not the quinolines like chloroquine or quinine.

<p>Fractional inhibition curve for the quinolines and artemisinin with FBS0701. Up to 10 uM FBS0701 does not change the low nM IC50 of artemisinin (filled square). In contrast for the quinolines 1, 5, 7.5 and 10 uM FBS0701 reduced the IC50 in an additive manner for chloroquine (empty circle), quinine (empty triangle) or quinidine (filled triangle). For each axis the concentration of drug in combination which equals the inhibition concentration 50% was divided by IC50 of drug alone.</p

Pharmacokinetics of FBS0701 in mice show high peak values.

<p>21 fed mice were given single oral dose at 100 mg/kg (filled triangle) and were sacrificed at indicated time with whole blood separated into erythrocytes and plasma. At least 200 microliters of plasma was analyzed for drug levels in the three mice at each time point. Error is standard deviation of the mean.</p

FBS0701 inhibits early stage not late stage gametocytes.

<p>Gametocyte cultures of <i>P. falciparum</i> NF54 strain were initiated at 0.5% asexual parasitemia and 4% hematocrit in 24 well culture plates. Gametocytes were visualized and counted on Day 18 with no drug (A) or 100 uM FBS0701 dosed in wells at Days 9–10 (B) or Days 14–15 (C) post-culture initiation. Dose dependent inhibition is more pronounced with early gametocyte stage I and II (day 9–10 dosing) than gametocyte late stages III-IV (day 14–15 dosing) (D). Gametocytes were inoculated at 0.5% parasitemia, resulting in gametocyte induction, followed by dosing of FBS0701 on indicated days. Gametocytes per 10 high power fields (excess of 1000 erythrocytes) in each of three replicate wells for each condition were counted on Day 18. Control wells had 7.4% mean gametocytes. Data expressed as percent inhibition of control. Standard deviation <u>of triplicate observations</u> for each of the three wells is shown.</p

Relief of allodynia depends upon the ß-common receptor (ßcR).

<p><b>A.</b> Both ketamine and ARA 290 did not prevent the development of allodynia in ßcR^−/− mice. <b>B.</b> However, the effect of ketamine on nociceptive pain is unchanged in ßcR^−/− animals (treatment effect, p<0.001).</p

FBS0701 treatment chelates intracellular erythrocytic iron to the same extent as deferiprone but unlike deferiprone, removes iron from erythrocytes.

<p>Aliquots 1–5×10⁶ erythrocytes from two separate healthy donors were stained in the dark with 0.125 µM calcein-AM for 15 minutes at 37°C. Cells were washed twice with PBS and allowed to rest for 10 minutes at 37°C in the dark. Flow cytometry on FACS- Calibur records fluorescent signal height of calcein on 40,000 cells. A. Calcein-AM fluorescence histograms of unstained (no calcein) erythrocytes (red) and calcein-stained erythrocytes (green) followed by incubation without (green) or with iron chelator L1 (blue) or FBS0701(yellow) show an increase in fluorescent signal intensity with iron chelator indicating displacement of labile iron from calcein. The L1 and FBS0701 peaks overlap. B. Washed erythrocytes after calcein-AM staining were incubated with PBS (green hatched bars), 100 µM L1(-deferiprone) (red dotted bars) or 100 µM FBS0701 (blue solid bars) chelator for 1 hr or 2 hr. Both chelators were able to enter erythrocyte and chelate iron once bound to calcein to increase MFI. C. Erythrocytes were incubated with PBS, 100 µM L1 (deferiprone) or 100 µM FBS7010 chelator for 1 hr and washed twice with PBS and incubated overnight in PBS. Erythrocytes were then washed, stained with calcein-AM and incubated with either L1 (red dotted bars) or FBS0701 (blue solid bars). The baseline calcein fluorescence in FBS0701 pre-treated cells was higher indicating a lower concentration of labile iron in these erythrocytes, while baseline MFI in cells incubated with deferiprone have is essentially unchanged. D. Change in Mean Fluorescent Intensity is shown for each of the conditions described in C. In the FBS0701 pre-treated cells, the addition of 100 µM of either chelator resulted in a change in MFI of 5 compared to a change of 15 in cells treated with either PBS or deferiprone. The data in B was performed in three replicates on RBCs from a single blood donor while C and D was performed using RBCsfrom two different donors each in triplicate and mean and standard deviation (SD) of the 6 data points is shown. Student’s t-test shows a statistical significant difference in the change in MFI for both iron chelators and PBS in B and C. In C, baseline FBS0701 MFI is higher than both PBS and deferiprone (p<0.05)and in D the change in MFI with FBS0701 after the overnight pre-treatment is less than with PBS and deferiprone (student’s t-test, p<0.005).</p

FBS0701 cures lethal <i>P. yoelii</i> in a single dose.

<p>Mice in groups of 4 were inoculated with 10 million lethal <i>P. yoelii</i> parasites by IP injection on day 0. A single oral dose of FBS0701 at 100 mg/kg given on day −7, day −1 and day +1 relative to infection. The survival curve shows complete protection with day +1 dosing and significant protection in animals pre-treated day −7 and day −1.</p

Ketamine and ARA 290 have similar effects on allodynia.

<p><b>A.</b> Treatment with both ketamine and ARA 290 prevented the full development of allodynia (treatment effect, p = 0.049 and p = 0.03, respectively). <b>B.</b> The effects of ketamine on acute nociceptive pain remained unchanged over time (treatment effect, p<0.001).</p

Neuropathic pain involves a pathway that utilizes the Innate Immune Receptor.

<p>Nerve injury results in microglial recruitment, increased expression of NMDAR, and proinflammatory cytokine production, ultimately resulting in allodynia. Activation of the innate immune receptor (IRR), e.g., by ARA 290, antagonizes this pathway. Ketamine also requires the IRR to reduce allodynia. This may be via a direct interaction with the IRR or alternatively, via modulation of intermediate processes that are upstream of the IRR. Additionally, ketamine interacts with NMDARs that mediate antinociception and psychomotor effects. ARA 290 does not interact with the NMDAR and therefore lacks these additional effects.</p

Ketamine and ARA 290 reduce inflammatory mediators in the spinal cord following sciatic nerve injury.

<p>One week post surgery, animals showed a marked elevation of CCL2 (panel <b>A</b>), Iba1 (panel <b>B</b>), and GFAP (panel <b>C</b>) compared to naïve controls. Both ketamine and ARA 290 significantly reduced the mRNA levels of these genes to a similar extent. *p<0.05 <i>versus</i> vehicle, #p<0.05 between ketamine and ARA 290 treatments, **p<0.05 <i>versus</i> naïve.</p