Description of the data and file structure

Premise: Tafenoquine (TQ) is an drug used to treat Plasmodium vivax malaria, however it can also cause hemolytic anemia in patients with glucose-6-phosphate dehydrogenase deficiency (G6PD-deficiency). pVCTQ is a polymer we have previously synthesized to treat P. vivax malaria without causing hemolytic anemia in those with G6PD-deficiency. We have synthesized a new polymer, pSVCTQ, which has a plasma-stable peptide linker. The goal was to create a polymer that reduces the drug release in plasma even further than the previous pVCTQ polymer, thereby reducing risk of hemotoxicity without decreasing drug activity. Each bolded section below corresponds to a different sheet in the excel file, and each sheet in the file corresponds to a figure in the manuscript or supplemental materials section and was used to create these figures.

Figure 2: pSVCTQ results in optimal TQ release, polymer biodistribution specificity to liver, and subcellular hepatocyte localization.

Liver and plasma concentrations of TQ after 25 mg/kg TQ equivalent dose of pSVCTQ or pVCTQ via intravenous (IV) route of administration and after 10 mg/kg TQ equivalent dose of pSVCTQ via SC route of administration or 10 mg/kg oral dose of free TQ. TQ was quantified in liver and plasma samples using LCMS/MS. Data is acquired via linear extraction. Graphing average data per timepoint along with corresponding standard deviation can recreate Figure 2 shown in the paper. This data highlights the differences between pSVCTQ, pVCTQ, and oral TQ, with pSVCTQ resulting in a high liver concentration and low plasma concentration compared to pVCTQ and oral TQ. Data shows TQ concentration for individual mice, average and standard deviation for each timepoint, AUC for the entire study, standard deviation for the AUC, Cmax (concentration at which max concentration is obtained in the PK profile), and Tmax (time at which Cmax is reached).

Figure 3. pSVCTQ has approximately twice the activity of oral TQ in the prophylactic P. berghei model.

Luciferase-expressing sporozoites are harvested from mosquitos. Mice are treated with either oral TQ or SC pSVCTQ, then inoculated with sporozoites. Bioluminescence of the sporozoites is recorded daily for 3 days using IVIS. From Day 5- Day 31, parasitemia is quantified using flow cytometry. Data shows the survival results of the pre-exposure prophylactic dose-response study. Mice were dosed with either 10mg/kg oral TQ or 5, 7.5, or 10 mg/kg TQ equivalent of pSVCTQ. No mice survived in the vehicle group. All mice survived in pSVCTQ 10mg/kg SC group. n=5 mice per treatment group. This data shows that pSVCTQ is twice as active as oral TQ at the same dose.

Figure 4. pSVCTQ reduces hemolytic toxicity in the humanized G6PD-deficiency mouse model.

Hemolytic anemia murine model: red blood cells are collected from a G6PD-deficient donor (huRBCs), then engrafted into NOD/SCID mice. Mice with >60% huRBCs at the end of the transfusion period (Day 0) are dosed with varying concentrations of oral TQ or SC pSVCTQ polymer solution. Data shows test article (either oral TQ or pSVCTQ), the dose of TQ given, the mouse ID, and the % hemolysis normalized to the vehicle control. Data is from a single experiment each. Historical oral TQ studies were done separately. The percent hemolysis was calculated for each treated animal from the percentage of huRBC present on Days 0 and 7, followed by normalization to the vehicle control group. Vehicle n =3, all other groups n=4. Experiment was replicated 3 times. Oral TQ hist. indicates historical data for a hemotoxicity study using an oral dose of TQ. This data shows reduce hemolytic toxicity from pSVCTQ compared to oral TQ.

Figure 5. pSVCTQ shows anti-hypnozoite activity in the P. cynomolgi/rhesus primary liver cell model.

Anti-hypnozoite assay: plate wells are seeded with hepatocytes on Day -2. On H0, cells are infected with P. cynomolgi sporozoites from Anopheles dirus salivary glands. Hypnozoites form over time and cells are treated with pSVCTQ solution on Days 4-7. On Day 8, cells are fixed and stained for imaging and hypnozoite detection. Data shows the different experiments, the drug concentration, % hypnozoite inhibition, and % toxicity to the hepatocytes. Each dose was replicated 3x. This data shows in vitro anti-hypnozoite activity with a great window between anti-hypnozoite activity and hepatocyte toxicity.

Fig. S9. Representative GPC trace of p(GalNAcMA-co-SVCTQMA) in LiBr-supplemented (0.1% w/v) DMF mobile phase at a flow rate of 1 mLmin-1.

This data can be used to show the GPC spectrum obtained for the pSVCTQ polymer.
This size-exclusion chromatography was performed using a Tosoh SEC TSK-GEL α-3000 and α-e4000 columns (Tosoh Bioscience, Montgomeryville, PA) connected in series to an Agilent 1200 Series Liquid Chromatography System (Santa Clara, CA), Wyatt Technology miniDAWN TREOS, 3 angle MALS light scattering instrument and Optilab TrEX, refractive index detector (Santa Barbara, CA). HPLC-grade DMF (0.1 weight% LiBr) was used as the mobile phase at a flow rate of 1 mL/min in a nonaqueous SEC system. Absolute molecular weight averages (Mn and Mw), polydispersity indices, and dn/dc were calculated using ASTRA software (Wyatt). This data shows the uniformity of pSVCTQ.

Fig. S11. PK of CTQ and OQTQ metabolites in liver, plasma, and urine.

Mouse pharmacokinetics of carboxy-TQ (CTQ) and 5,6 orthoquinone-TQ (OQTQ) after subcutaneous dose of pSVCTQ. Time-course metabolite concentrations in liver, plasma, and urine were determined using LC-MS/MS. Each value represents the mean ± standard deviation (n = 3–4). This was done at a dose of 10 mg/kg TQ equivalent. This data shows the PK profiles of two important metabolites for TQ. Not all timepoints had urine samples collected as some mice did not void their bladder. Liver is in ng/g liver; Plasma is in ng/mL plasma; Urine is in ng/mL urine.

Fig. S13. Efficacy of SVCTQ polymer and oral TQ in P. berghei causal prophylaxis model during liver-stage of infection.

Mice were given a single dose of either TQ (10 mg/kg, PO) or SVCTQ polymer (5, 7.5, or 10 mg/kg, SC), or vehicle (DPBS, SC) on Day −1 with respect to sporozoite infection. IVIS imaging was completed on Days 1 through 3. IVIS signal, measured in total flux of photons, measured at each timepoint is correlated with the levels of luciferase-expressing P. berghei in the liver. Lack of IVIS signal indicates a suppression of sporozoite proliferation in the liver. The vehicle had a high amount of signal while the treatment groups had zero. This indicates very little sporozoite proliferation during these timepoints for the treatment groups.
Blood stage parasitemia results over 31 days for each individual mouse are shown under the parasitemia plot data. mouse blood was analyzed for blood stage infections by quantitation of malaria parasites in erythrocytes via flow cytometry. This data shows the individual mouse and dose, as well as % parasitemia at each timepoint. 0/5 vehicle-treated mice survived; 2/5 oral TQ-treated mice survived; 2/5 5mg/kg polymer-treated mice survived; 4/5 7.5mg/kg polymer-treated mice survived; 5/5 10 mg/kg polymer-treated mice survived.
All flow cytometric analyses were carried out with an FC500 MPL flow cytometer (Beckman Coulter, Fullerton, CA) for conducting 5-color analysis from either single or dual laser excitation. Infected erythrocytes, uninfected erythrocytes, and leukocytes were gated on logarithmic forward/side dot plots. Cells were analyzed at an average rate of 2000–3000 erythrocytes per second. Filters were placed before the green (FL-1), and red (FL-2) photomultiplier tubes (PMT) such that the green PMT registered fluorescence emission between 520 and 555 nm, and the red PMT measured emission greater than 580 nm.

Fig. S14. Reticulocyte, hematocrit, and spleen weight following SVCTQ polymer administration in a hemotoxicity model.

This sheet has data for the following: % peripheral blood levels of mouse reticulocytes on day 0, 4, and 7 to show kinetics of mouse reticulocyte production, body weight on day 0 and 7, spleen Weight on Day 7 and normalized to vehicle, and percent Hematocrit (HCT) Levels on Day 7 for each mouse in the Oral TQ, SC pSVCTQ, and vehicle treatment groups. Reticulocytes were measured using flow cytometry. This data is supplemental to show further markers of hemolytic toxicity.

Fig. S15. PK of TQ in liver after SVCTQ, SVATQ or Tri-antennary GalNAc polymer administration.

Mouse pharmacokinetics of TQ after SC administration of the p(GalNAc-co-SVCTQ) (pSVCTQ), p(GalNAc-co-SVATQ) (pSVATQ), p(GalNAc-co-MSEMA-co-SVCTQ) (Low GalNAc pSVCTQ), and the p(Tri-antennary GalNAc-b-(GMA-co-SVATQ) (Trigal pSVATQ) polymers at 25mg/kg and 10mg/kg doses of TQ equivalent. TQ concentrations in liver over time were determined using LC-MS/MS. Each value represents the mean ± standard deviation (n = 3). Liver is in ng/g liver, plasma is in ng/mL plasma. Data shows TQ concentration for individual mice, average and standard deviation for each timepoint, AUC for the entire study, standard deviation for the AUC, Cmax (concentration at which max concentration is obtained in the PK profile), and Tmax (time at which Cmax is reached). This data shows a few alternative polymer designs we have completed which may be used in the future to further optimize the TQ-based polymer.

Please see manuscript when interpreting this data.

Abbreviations:
PK: pharmacokinetics
TQ: tafenoquine
AUC: area under the curve
SD: standard deviation
Cmax: Maximum concentration obtained in the PK profile
Tmax: time at which Cmax is reached
PO: per os, indicating oral dose administration
SC: subcutaneous administration
IV: Intravenous
mpk: mg/kg