Hydroxychloroquine prophylaxis and treatment is ineffective in macaque and hamster SARS-CoV-2 disease models

We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in 2 animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg given daily) did not significantly benefit clinical outcome, nor did it reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment, neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these 2 preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19.

Syrian hamster study design. The hamster study was designed with two arms, prophylaxis and therapeutic. Hamsters were divided into groups for either prophylaxis treatments or therapeutic treatments (n=6 per group). Two groups were treated one time with either a 6.5 mg/kg or 50 mg/kg 24 hours prior to infection for the prophylaxis arm. There were two therapeutic groups, one group received 6.5mg/kg while a second received 50mg/kg. Treatments began 1-hour postinfection and were performed every 24 hours on days 1, 2 and 3 post-infection. A final group consisted of vehicle control animals that received the same volume of PBS as the prophylactic and therapeutic groups. All groups were infected intranasally with 100ul of 1x10^5 median tissue culture infectious dose (TCID50) of SARS-CoV-2 (50 uL/nare). Animals were euthanized and samples collected at day 4 post-infection. All procedures were performed on anesthetized animals. Swabs (oral, rectal) were collected at days 2 and 4, and lung tissues were collected at necropsy on day 4 post-infection for pathology and virology.
Rhesus macaque study design. The study also consisted of two arms, prophylaxis and treatment (Fig. 1A). Animals were anesthetized for all procedures. For the prophylaxis arm, 10 healthy rhesus macaques (all male; 4.9 -5.6 kg in weight) were randomly divided into vehicle control (n=5) and HCQ prophylaxis (n=5) groups. Animals were treated with either vehicle (PBS) or HCQ (6.5mg/kg in PBS) three times one week apart (days -9, day -2 and day 5) by oral gavage.
In the second arm, 10 healthy rhesus macaques (all male; 5.7 -7.3 kg in weight) were randomly divided into vehicle control (n=5) and HCQ treatment (n=5) group. Animals were treated with either vehicle (PBS) or HCQ (6.5 mg/kg in PBS) starting 12 hours post-infection followed by treatment at 18, 36, 60, 84 and 108 hours post-infection by oral gavage. All animals were infected on day 0 with a total dose of 2.8 x10^6 TCID50 of SARS-CoV-2 by a combination of four routes (intratracheal, oral, intranasal and ocular) as established and recently used in a drug efficacy study. Animals were monitored at least twice daily using an established scoring sheet by the same person, who was blinded to the group assignments, throughout the study (1). Physical examinations were performed on days -9, -2, 0, 1, 3, 5, and 7 and included a clinical evaluation, radiographs, venous blood draw, and swabs (oral, nasal and rectal). Bronchoalveolar lavage (BAL) was performed on day 3, 5 and 7 post infection. The study endpoint was day 7. Following euthanasia, necropsies were performed, and gross lung lesions were scored by a board-certified veterinary pathologist blinded to the group assignment.
Liquid chromatography and mass spectrometry. LCMS grade water, methanol, acetonitrile and formic acid were purchased through Fisher Scientific. All synthetic standards for molecular analysis were purchased from Santa Cruz Biotechnology Inc. Levels of HCQ and secondary metabolites were determined using methodology established previously with modifications (2).
Plasma and cleared lung homogenates were gamma-irradiated (2 megarads) prior to removal from biocontainment according to IBC-approved protocol. Plasma samples were prepared for small molecule analysis by diluting a 25 µL aliquot with 100 µL of 0.1% formic acid and 1 mL of acetonitrile on ice. Clarified lung homogenate samples were prepared by adding 1 mL of acetonitrile to a 250 µL aliquot of lung homogenate. Samples were vortexed and incubated at -20 o C for 2 hrs. Samples were centrifuged at 16,000 x g and 4 o C for 20 min. The clarified supernatants (1 mL) were recovered and taken to dryness in a Savant™ DNA120 SpeedVac™ concentrator (Thermo Fisher). Samples were resuspended in 100 µL of 50% methanol, 50% water (v/v) and centrifuged as before. The supernatant was taken to a sample vial for LCMS analysis. Samples were separated by reverse phase chromatography on a Sciex ExionLC™ AC system. Samples were injected onto a Waters Atlantis T3 column (100Å, 3 µm, 3 mm X 100 mm) and eluted using a binary gradient from 25 % methanol, 0.1 % formic acid to 100 % methanol formic acid over 4 min. Analytes were measured using a Sciex 5500 QTRAP® mass spectrometer in positive mode. Multiple reaction monitoring was performed using previously established signal pairs for each analyte and signal fidelity was confirmed by collecting triggered product ion spectra and comparing back to spectra of synthetically pure standards (2). All analytes were quantified against an 8-point calibration curve of the respective synthetic standard prepared in the target matrix and processed in the same manner as experimental samples. Limits of quantification in plasma for all metabolites was 0.5 ng/mL. Limit of quantification in lung homogenate for all metabolites was 6 ng/mL. Apparent and sample data were filtered prior to weight normalization.
Thoracic radiographs. Ventro-dorsal and right/left lateral radiographs were taken of nonhuman primates on clinical exam days and scored for the presence of pulmonary infiltrates by two clinical veterinarians according to a standard scoring system (0: normal; 1: mild interstitial pulmonary infiltrates; 2: moderate pulmonary infiltrates perhaps with partial cardiac border effacement and small areas of pulmonary consolidation; 3: serious interstitial infiltrates, alveolar patterns and air bronchograms). Individual lobes were scored. Scores from the lobes were then totaled and recorded per animal per day.
Virus load. RNA was extracted from swabs and BAL using the QIAamp Viral RNA kit (Qiagen) according to the manufacturer's instructions. Tissues were homogenized in RLT buffer and RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions. For detection of viral RNA, 5 µl RNA was used in a one-step real-time RT-PCR E assay (3) using the Rotor-Gene probe kit (Qiagen) according to instructions of the manufacturer.
In each run, standard dilutions of RNA standards counted by droplet digital PCR were run in parallel, to calculate copy numbers in the samples.

Virus titration.
Virus isolation was performed on lung tissues by homogenizing the tissue in 1 mL DMEM using a TissueLyser (Qiagen) and inoculating Vero E6 cells in a 24 well plate with 250 µL of cleared and a 1:10 dilution of the homogenate. One hour after inoculation of cells, the inoculum was removed and replaced with 500 µL DMEM (Sigma-Aldrich) supplemented with 2% fetal bovine serum, 1 mM L-glutamine, 50 U/mL penicillin and 50 µg/mL streptomycin. Six days after inoculation, cytopathogenic effect was scored and the TCID50 was calculated.
Histopathology and immunohistochemistry. Histopathology and immunohistochemistry were performed on rhesus macaque tissues. After fixation for a minimum of 7 days in 10 % neutral-eosin (H&E). Stained slides were analyzed by a board-certified veterinary pathologist.
Statistical analyses. Statistical analysis was performed in Prism 8 (GraphPad).

References.
(1) D.L. Brining et al., Thoracic radiography as a refinement methodology for the study of H1N1   Reduced appetite, ruffled fur, slow, increased irregular abdominal respirations Score: 15/13/10/15 HCQ, hydroxychloroquine; PS, prophylaxis; TS, treatment; The light gray underlined rows indicate animals that were administered drug either as prophylaxis or treatment.  TS  PBS  <5  <LOD  <LOD  <LOD  <LOD  ND  ND  ND  ND   H27  TS  PBS  <5  <LOD  <LOD  <LOD  <LOD  ND  ND  ND  ND   H28  TS  PBS  <5  <LOD  <LOD  <LOD  <LOD  ND  ND  ND  ND   H29  TS  PBS  <5  <LOD  <LOD  <LOD  <LOD  ND  ND  ND  ND   H30  TS  PBS  <5  <LOD  <LOD  <LOD  <LOD  ND  ND  ND  ND PS, prophylaxis; TS, treatment; HCQ, hydroxychloroquine; DCQ, desethylchloroquine; BDCQ, bisethylchloroquine; DHCQ, desethylhydroxychloroquine; Quantification of HCQ and secondary metabolites in lung sections from hamsters at day 4 post infection reported as total ng in the analyzed sample. Samples with a mass greater than 5 mg were weight normalized to indicate ng/mg of tissue. <LOQ: below limit of quantification but above limit of detection. <LOD: below limit of detection. *Samples from H3 and H17 were not available for drug level analysis. ND: Not Done due to insufficient sample. Radiograph scores -Therapeutic (TS). T tests were used to evaluate results, no significant difference was found between groups.