pa300 croi 2016 final · 2020. 4. 19. ·...
TRANSCRIPT
This project was funded by NIH grant U19MH081835 and R01NS04237. We thank Synergenics, LLC for providing the PA300 and Wedgewood Pharmacy for compounding PA300 into syrup and for placebo.
SIV-‐Associated Pathogenesis Modula6on with Macrophage Targeted MGBG
Background
1 Department of Biology, Boston College, Chestnut Hill, MA, USA; 2 Pathologica LLC, Burlingame, CA, USA; 3 Department of Biomedical Sciences, SecCon of Anatomic Pathology, Cornell University College of Veterinary Medicine, Ithaca, NY, USA; 4 Department of Laboratory Medicine, Medicine
and Pathology, AIDS & Cancer Resource Center, San Francisco General Hospital, San Francisco, CA, USA.
Tricia H. Burdo1, Joshua Walker1, Hua Xu2, Andrew D. Miller3, Michael S. McGrath4, and Kenneth C. Williams1
Figure 1
Figure 2
Methods The role of macrophages HIV infecRon and AIDS pathogenesis is controversial. Despite this, macrophages are consistently idenRfied as key cells in HIV-‐associated comorbidiRes including cardiovascular disease and HIV-‐associated neurocogniRve disorders (HAND). This study used a novel oral form of a polyamine biosynthesis inhibitor MGBG (PA300), that exclusively targets acRvated and infected macrophages, in an SIV infecRon associated pathogenesis study. PA300 is selecRvely concentrated in myeloid cells through mechanisms consistent with acRve transport by the polyamine transporter. In vitro studies show it decreases HIV expression in monocytes and macrophages in a dose dependent manner and viral life cycle mapping show that MGBG inhibits DNA integraRon into the cellular DNA in these cells (Jin, McGrath, Xu, 2015, J Virology, 89:22, 11176-‐11189). In vitro and in vivo data demonstrate the selecRve nature of PA300 targeRng myeloid cells, blocking the development of AIDS, SIV encephaliRs, and diminishing the effects of AIDS on cardiovascular disease. Histopathological and CBC data demonstrate PA300 treatment has no Rssue toxicology. PA300 treatment resulted in the majority of animals not developing AIDS, decreased inflammaRon in the CNS, cardiac Rssues and aorta. There was a decreased number of inflammatory macrophages in the CNS, and decreased cardiac fibrosis, caroRd artery inRma-‐media thickness and cardiomyocyte damage. These data point to the potenRal use of PA300 as an adjuncRve therapy with cART for acute and chronic HIV infecRon
Two studies were performed using SIVmac251-‐infected CD8-‐depleted rhesus macaques. The first was a 30 day pK study (0, 3, 10 and 30mg/kg/day PA300 n=2/group)(PA300 provided by Pathologica, LLC; formulated by Wedgewood Pharmacy, Swedesboro, NJ) and the second was a blinded longitudinal study with daily dosing of 30mg/kg for 9 weeks (untreated, n=6 and PA300 n=9). Both studies began PA300 at 21 days post-‐infecRon (dpi). Control and treated animals were grouped and sacrificed with AIDS or by 84 dpi. Blood studies were performed longitudinally. Clinical blood chemistries and full SIV necropsies were performed. IHC analysis including quanRtaRve analysis of CNS and cardiac Rssues, enumeraRon of macrophage infiltraRon, analysis of cardiac fibrosis, aorRc inRma media thickness, and cardiomyocyte damage.
PA300 down regulates CD16+ monocytes in a dose dependent manner. PA300 down regulates CD16 on CD14+ monocytes ager 3 days of treatment. Live/dead staining demonstrated cell toxicity.
PA300 treatment (daily dose of 30mg/kg star6ng at dpi 21) stops the development of SIVE and lowers the incidence of AIDS in SIV-‐infected rhesus macaques. 4 of 8 (50%) placebo treated SIV-‐infected animals developed SIVE and 7 out of 8 (87.5%) developed AIDS defining lesions. In the PA300 treatment group, 0 out of 11 (0%) animals developed SIVE and only 4 of 11 (36%) animals had AIDS defining lesions. Animals in groups 1-‐3 were sacrificed in pairs with 1 placebo and 2 PA300 treated animals upon the development of disease of the placebo animal. Animals in groups 4, 5 and BQ were Rmed sacrificed. BQ= Bioqual first pk study, SIVE= SIV encephaliRs, No AIDS= no AIDS defining lesions, AIDS= acquired immunodeficiency syndrome.
Table 1
Acknowledgments
Conclusions • A biologically effecRve dose of PA300 (0.7uM) was achieved in animals that received 30mg/
kg)(oral route, daily dose) where drug levels greater than 0.7uM found in plasma, brain, liver and kidney.
• Pre-‐clinical outcome data and histopathologic analysis demonstrated no drug-‐related toxiciRes with any drug dose.
• PA300 Rx resulted in: • Decreased CD68+ and CD163+ macrophages in the brain, and SIVE lesions • Decreased caroRd artery inRma-‐media thickness and inflammaRon. • Decreased inflammaRon in leg ventricle Rssue. • Decreased levels of leg ventricle fibrosis (as measured by collagen). • Decreased numbers of CD68+, CD163+, MAC387+ and CD206+ macrophages
in leg ventricle.
PA300 reaches an effec6ve level in plasma, brain, liver, spleen, kidney and lymph nodes. An effecRve dose of PA300 (0.7uM) was achieved in animals that received 30mg/kg daily by an oral route. Drug levels greater than 0.7uM found in plasma (leg), brain, liver and kidney (right). PA300 was found in brain but not in the CSF at any dose.
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PA300 treatment decreased the numbers of CD163+ and CD68+ inflammatory macrophages in the brain. Brain secRons from Placebo or PA300 treated animals were stained anRbodies against either (A-‐C) CD163 or (D-‐F) CD68. Eight secRons of each frontal cortex (A, D), parietal cortex (B, E) and occipital cortex (C, F) were assessed for the number of macrophages per mm2 of Rssue. Data are presented as the mean and standard error of the mean. In each cortex of the brain examined, PA300 treatment decreased the amount of both CD68 and CD163 macrophages. The number of MAC387+ cells was not significantly changed, but was elevated in animals with SIVE. *= P<0.05.
Figure 5 SIV+ Placebo Treated SIV+ PA300 Treated
inRma
inRma
media
media
Internal elasRc lamina
Internal elasRc lamina
Caro6d artery in6ma media thickness (cIMT) is decreased with PA300 treatment. The inRma media thickness was measured in Placebo and PA300 treated SIV infected rhesus macaques. The mean caroRd artery inRma media thickness was significantly decreased with PA300 treatment. cIMT is used as a diagnosRc measurement for subclinical atheroscelorsis. *= P <0.05
PA300 significantly decreases the number of CD68+, CD163+, MAC387+ and CD206+ macrophages in the le_ ventricles. Eight secRons from all animals were quanRtated for the number of CD68+ (A), CD163+ (B), MAC387+ (C) and CD206+ (D) macrophages per mm2 of leg ventricle Rssue. The mean and standard error of the mean are shown. PA300 treatment decreased the amount macrophages present. *= P<0.05, **= P<0.01. There were no staRsRcally significant differences in number of macrophages in the caroRd arteries.
Figure 7
SIV+ Placebo Treated SIV+ PA300 Treated PA300 decreases the percentage of collagen (fibrosis) in the le_ ventricle. Ventricle secRons were stained with Mason’s trichrome and collagen (blue) was quanRtated as a measure of cardiac fibrosis. PA300 treatment significantly decrease the percent of collagen in the ventricle compared to placebo SIV-‐infected macaques. **= P <0.01.
Figure 6
Table 2
Cardiac pathology was decreased with PA300 treatment. Both caroRd artery and leg ventricle pathology was decreased with PA300 treatment. NA= not available. CaroRd arteries were not available for assessment in the Bioqual (BQ) pk study.
Figure 3
PA300 treatment blocks development of SIVE. 4 of 8 SIV-‐infected placebo controls developed SIVE as shown (leg). The insert shows an SIVE lesion with a mulRnucleated giant cell (MNGC) (arrow). None of the PA300 Rx (0/11) developed SIVE (right).
Figure 4
Placebo: 4/8 SIVE 7/8 AIDS 1/8 no AIDS
PA300: 0/11 SIVE 4/11 AIDS 7/11 no AIDS
PA300 treatment blocks the development of AIDS (36%) and SIVE (0%)!
AIDS defining lesions