DOI: 10.5061/dryad.3bk3j9krr
title: Liver-targeted polymeric prodrugs delivered subcutaneously improve tafenoquine therapeutic window for malaria radical cure

description: Approximately 3.3 billion people live with the threat of Plasmodium vivax
 malaria. Infection can result in liver-localized hypnozoites, which when
 reactivated cause relapsing malaria. This is the first demonstration of an
 enzyme-cleavable polymeric prodrug of tafenoquine (TQ) that addresses key
 requirements for a mass administration, eradication campaign: excellent
 subcutaneous bioavailability, complete parasite control after a single
 dose, improved therapeutic window compared to the parent oral drug, and
 low Cost of Goods Sold (COGS) at less than $1.50/dose. Liver-targeting and
 subcutaneous dosing resulted in improved liver:plasma exposure profiles,
 with increased efficacy and reduced Glucose 6-Phosphate Dehydrogenase
 (G6PD)-dependent hemotoxicity in validated preclinical models. A COGS and
 manufacturability analysis demonstrated global scalability, affordability,
 and the ability to redesign this fully synthetic polymeric prodrug
 specifically to increase global equity and access. Taken together, this
 polymer prodrug platform is a candidate for evaluation in human patients
 and shows potential for P. vivax eradication campaigns.
description: # Liver-Targeted Polymeric Prodrugs Delivered Subcutaneously Improve
 Tafenoquine Therapeutic Window for Malaria Radical Cure ## 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 &gt;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