Biological Evaluation of Compounds

 

A p.o. P.Berghei mouse study at 50 mg/kg revealed no efficacy of OSM-S-35 (ZYH 3-1),OSM-S-5 (TCMDC-123812) or OSM-S-6 (TCMDC-123794) relative to control. Experiments carried out at Swiss TPH. Experiment start 23/04/12.



Method Overview:
"This protocol is performed in the group of Dr Sergio Wittlin at Swiss TPH (Unit of Prof. Reto Brun), for assessing compound efficacy against the Plasmodium berghei GFP ANKA strain in-vivo. Mice are infected intravenously with parasitized red blood cells on day 0 (2 x 107 parasitized erythrocytes per ml). Experimental mice are generally treated at 4, 24, 48, and 72 hours post-infection with an oral dose of the compound (4-day test by Peters) and are compared to an infected control group for reduction in parasitaemia on day 4 (96 hours post-infection) in % and for mean survival (monitored up to 30 days post-infection). A compound is considered curative, if the animal survives to day 30 after infection with no detectable parasites. Other delivery route (intravenous, intraperitoneal, subcutaneous) and dosing regimen (e.g. single dose) are possible.

Percent activities below 40% are regarded as inactive.

To put the data into context, a new lead optimization project should typically show an oral ED50 <50mg/kg while a development candidate will typically have an oral ED90 <10mg/kg"

The raw data in excel format:

Biological testing of the 2nd round of compounds reported on 05/05/12 by James S. Pham from the University of Melbourne.

Visual summary of results and compound structures:


Methods Overview:
"Compounds were tested for inhibition of Plasmodium falciparum growth using a SYBR green I fluorescence based assay. Dose response curves were generated comprising of 8 points using a 2-fold serial dilution for the maximum final concentration of 1000 nM. The lowest concentration tested was 7.81 nM."

Raw Data originally posted on Google+ along with a detailed method description and report.

Raw data and graphs in excel format:


Compounds tested:
PMY 12-5, PMY 27-2, PMY 31-5, PMY 34-1, ZYH 3-1, ZYH 5-1, ZYH 6-1/6-2, ZYH 7-2, ZYH 10-2 A, ZYH 10-2 B, ZYH 12-1/12-2, ZYH 15-1, ZYH 16-1, ZYH 17-1, ZYH 18-1, ZYH 19-1, ZYH 22-3, ZYH 23-1

Analysis of the same compounds:
Second Round of Evaluation by GSK Tres Cantos
Second Round of Evaluation by the Avery Lab

Results largely consistent with above analyses.

Data acquired by Karen White External Projects Coordinator, Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia

Compounds examined:

Data (xls):

The 0-1.6 µg/mL window means that the solubility was less than the lowest concentration tested which in this assay is 1.6 µg/mL.

Conclusion: That some compounds displayed low degradation at the expense of low solubility.

Data from Sandra Duffy, Griffith University
Received April 25th 2012

Four compounds from the arylpyrrole series tested in a late stage anti-gametocyte imaging assay.

Relevant compounds:


Method: The assay involves the use of a Pfs16-GFP transgenic NF54 Plasmodium falciparum strain kindly provided by Dr David Fidock (Columbia University).

Compound is added to 384 well imaging plates containing 20,000 highly synchronous late stage IV gametocytes. The plates are incubated for 72 hours in standard incubation conditions (5%CO2, 5%O2, 37oC, 5% humidity). After incubation the viability marker MitoTracker Red CM-H 2 XRos is added to the wells. The plates are then imaged on the OPERA confocal high-throughput imaging system. The images obtained are then analysed using an algorithm for determining the number of viable gametocytes still remaining in the compound treated wells based on viability marker fluorescent intensity and the morphology of the GFP expressing parasite.

The number of gametocytes per well in this assay is the same number of parasites used in the asexual imaging assay for 3D7 and K1.

Results: PMY10-2 has no significant activity whilst PMY14-1 has activity comparable to that in the asexual assay. (MHT: Others?)

Raw data:

Caveat: It has to be noted that the compounds have been in storage for a while and the plates defrosted several times for screening. The inactive compound could have lost asexual activity at the same time due to some instability. This was not tested.

Assay performed by Essen Bioscience 17th April 2012

Relevant compounds:


Methods overview
Compounds were tested for inhibition of the human ether a go-go related gene (hERG) K+ channel using IonWorks patch clamp electrophysiology. 8-Point concentration-response curves were generated using 3-fold serial dilutions from the maximum final assay concentration shown in this spreadsheet.

Image of results:


(legend: Compound (ID, number in assay), pIC50 (value, 95% CI), IC50 in micromolar(value, 95% CI), Curve profile (slope, [max] in micromolar, cell count, QC, notes). For full explanations, see notes in spreadsheet linked above)

Graphs of outputs:

OSM-S-35 (ZYH 3):
OSM-S-5 (Original GSK, PMY 10):
Quinidine:

In each graph the log of molar concentration of test compound is plotted against the effect of the compound on the hERG current, expressed as a percentage of the pre-compound signal. In this experiment an average of 9±3% signal decay was observed in the time- and vehicle (DMSO) control. Data are normalised for this vehicle-response such that a value of 100% = no drug effect. The smaller grey symbols represent data from individual cells. The larger blue points show the mean data at each test concentration. The line of best fit (4 parameter logistic equation) is generated from the individual cell data points. Outlier data points (boxed symbols) were manually excluded from the curve fit. No more than two per curve were excluded, other than where compounds were visibly out of solution.

Details of method
Electrophysiological recordings were made from a Chinese Hamster Lung cell line stably expressing the full length hERG channel. Single cell ionic currents were measured in the perforated patch clamp configuration (100 μg ml-1) amphoterocin) at room temperature (21-230C) using an IonWorks Quattro instrument. The internal solution contained (mM): 140 KCl, 1 MgCl2, 1 EGTA, 20 HEPES and was buffered to pH 7.3. The external solution contained (mM): 138 NaCl, 2.7 KCl, 0.9 CaCl2, 0.5 MgCl2, 8 Na2HPO4, 1.5 KH2PO4 also buffered to pH7.3. Cells were clamped at a holding potential of -70mV for 30s and then stepped to +40mV for 1s. This was followed by a hyperpolarising step of 1s to -30mV to evoke the hERG tail current. This sequence was repeated 5 times at a frequency of 0.25Hz. Currents were measured from the tail step at the 5th pulse, and referenced to the holding current. Compounds were then incubated for 6-7 minutes prior to a second measurement of the hERG signal using an identical pulse train.



The following QC conditions were applied:

(1) Individual cells with any of the following properties were excluded from subsequent analysis: (1) seal resistances <50MOhms (2) hERG currents <150pA (3) seal resistances that changed by >50% during the experiment

(2) A minimum of 17 cells were required for each pIC50 curve fits

(3) pIC50 curve fits with a 95% confidence limit of > ±0.5 log were failed

(4) Entire assay plates in which the pIC50 of the standard compound (quinidine) was outside of the normal range [5.6-6.3] were failed.