Prodrugs extend the half life and potency of Cabotegravir Tanmay A. Kulkarni1,2*†, Aditya N. Bade2*, Brady Sillman2, Bhagya Laxmi Dyavar Shetty2, Melinda Wojtkiewicz2, JoEllyn McMillan2, Benson Edagwa2 and Howard E. Gendelman1,2 1Department of Pharmaceutical Sciences, 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198. Poster# 489 *Authors contributed equally, †Presenting author
Abstract Pharmacokinetics Antiretroviral activity of NM2CAB
BACKGROUND Prevention of new infections, reduction in transmission and management of chronic infection characterize once a month dosing of cabotegravir (CAB) in its current formulation (CAB LA, referred henceforth as NCAB). We previously demonstrated that the potency, bioavailability and tissue distribution of CAB could be improved by A increasing drug lipophilicity and hydrophilicity.A first generation prodrug (MCAB) nanoformulation (NMCAB) extended the protein adjusted IC90 (PA-IC90) up to 3 months in rhesus macaques after a single 45 mg/kg CAB equivalent intramuscular (IM) injection. We now report modifications of CAB designed to further reduce dosing frequency while improving drug bioavailability. METHODS A second generation CAB prodrug (M2CAB) was synthesized through bioreversible acylation. Poloxamer 407 nanoformulations (abbreviated NM2CAB) were prepared by high-pressure homogenization. Electron microscopy was used to evaluate particle shape and size. Human monocyte derived macrophages (MDM) and CD4+ T cells were the biological platforms employed to measure drug uptake, retention and release. Antiretroviral activity was examined by culture fluid assay for reverse transcriptase activity and cell- based HIV-1p24 antigens recorded by immunohistochemistry. Pharmacokinetic evaluation was performed in humanized NOD/scid-IL-2Rγc null (NSG) mice and rhesus macaques. RESULTS NM2CAB exhibited uniform particle size of 300-350 nm, narrow polydispersity index (PDI) of 0.2, a negative zeta potential and a high drug loading capacity (> 80%). Physical and thermal stability of NM2CAB at 25°C was observed with no particle agglomeration. NM2CAB was retained in MDM (levels of 8 nmol/106 cells) at one month. NM2CAB attenuation of viral replication was observed to 30 days compared to 15 and 1 day for NMCAB and NCAB, respectively. After a single 45 mg/kg CAB equivalent IM injection of NM2CAB in mice, plasma CAB levels were > 4 times the PA-IC90 (664 ng/ml) for > 8 months compared to 2.5 and 1 month for NMCAB and NCAB, respectively and replicated in rhesus macaques in ongoing studies. CONCLUSIONS The hydrophobicity and sustained slow hydrolysis of the M2CAB prodrug facilitate NM2CAB to harness the injection site as a primary drug depot as well as macrophages and other tissues as secondary drug depots for months. This can potentially reduce frequent dosing and injection volume improving patient adherence to B antiretroviral therapy. Days 1 5 10 15 20 25 30
Results HIV-1
Synthesis of M2CAB A NCAB B C + DIEA DMF, 25ºC Acyl CAB chloride M2CAB NMCAB D
NM2CAB E F G Figure 3. Pharmacokinetics of NM2CAB. Plasma CAB levels determined over eight months after a single 45 mg/kg CAB equivalent IM injection of NM2CAB, NMCAB and NCAB (a CAB LA equivalent formulation). This study parallels results seen in rhesus macaques and remains ongoing.
In vitro characterization of NM2CAB Control A B C
Figure 5. Antiretroviral activity of NM2CAB. (A) MDM were treated with 100 μM NM2CAB, NMCAB or NCAB for 8 hrs (drug loading) at the time points listed. MDM were challenged with 0.1 MOI HIV-1ADA after drug loading. Ten days after viral challenge, reverse transcriptase (RT) activity was measured in culture medium and (B) cells were stained for HIV-1 p24 antigen.
Figure 1. Synthesis and characterization of M2CAB (A) Synthesis scheme (B) FTIR spectroscopy of CAB and M2CAB (C) Drug concentration for 50% HIV inhibition (IC50) of CAB (20.8 nM), MCAB (7.1 nM) and M2CAB (6.5 nM) in human MDM (D) 1H-NMR spectra of CAB and M2CAB (E) Depletion of M2CAB into active CAB in physiological conditions (plasma of various species) at 37 ºC (F) CAB formation (G) Decreased aqueous solubility of M2CAB at room temperature is shown Conclusions
Synthesis and characterization of M2CAB nanoformulation (NM2CAB) NM2CAB B • demonstrated up to 10-fold improvement in pharmacokinetics over unmodified CAB nanoformulation. A C • was found safe for administration in animals and no cytotoxic effects were seen in vitro. • has excellent scalability owing to its simple design and optimized straight forward synthesis. • showed stability over 100 days in terms of particle size, charge and drug content integrity. • is potent with sustained antiretroviral activity. • is retained and released slowly and effectively by macrophages.
D Acknowledgements
This research was supported by the University of Nebraska Foundation; the Vice Chancellor for Research Office, University of Nebraska Medical Center; and National Institutes of Health grants, P01 DA028555,R01 NS36126,P01 D E NS31492, 2R01 NS034239,P01 MH64570,P01 NS 43985, 1 R56 A1 138613-01A1, 3P30 MH062261,P30 AI078498, 1R24 OD018546, and R01 AG043540.
Figure 4. In vitro characterization of NM2CAB (A) Uptake and (B) Retention of NM2CAB in MDM compared with NMCAB and NCAB. For uptake, drug concentration was determined in MDM treated with 100 μM NM2CAB, NMCAB or NCAB for 2-24 hours. Cell drug retention was determined after treatment of MDM for 8 hrs with 100 μM Figure 2. Synthesis and characterization of NM2CAB (A) Synthesis of nanoformulated M2CAB (NM2CAB) (B) SEM of NM2CAB shows rod shaped nanoparticles (C)Stability NM2CAB, NMCAB or NCAB, followed by replacement with fresh medium without drug. Drug concentration determined from 1-30 days. (C) CAB release was determined of NM2CAB in terms of particle size, polydispersity index and zeta potential over 100 days at 25 ºC (D) MTT assay shows no cytotoxicity of NM2CAB in MDM at various for 30 days from MDM treated with 100 μM NM2CAB, NMCAB and NCAB. (D) TEM of MDM at the end of 30 days after treatment for 8 hrs with 100 μM NM2CAB, NMCAB, concentrations (E) X-ray diffraction pattern illustrates that crystallinity of M2CAB is retained in NM2CAB or NCAB. The cells exposed to NM2CAB showed nanocrystals (red arrowheads) while NMCAB and NCAB showed no drug crystals.