Journal critique of the usage of prodrug nanoparticles for a long-acting depot of antiretroviral drugs

for HIV treatment

Emily Thomas1,*, Hugo Pontes1,*
1
Department of Chemical Engineering, University of Washington, Seattle WA
*Authors contributed equally

Purpose and Context

Currently, human immunodeficiency virus (HIV) is treated with antiretroviral (ARV) therapy, a daily ingestion of
HIV medications to reduce viral load. The monotony leaves people open to “pill fatigue” [1]. Since continual
compliance is vital to maintaining a low viral load, nonadherence is dangerous to HIV patients. It can allow the
virus to mutate into a drug resistant strain. In developed countries, people have access to a plethora of drug
combinations. However, most HIV positive people live in developing countries in sub-Saharan Africa, where
they have access to as few as three generic drug combinations [2]. This leaves very few ways to combat drug
resistant strains since there are few limited drugs options available.  Even in countries where there is access to
a wide variety of ARVs, noncompliance is dangerous. Having a drug resistant strain could mean complete, and
fatal, treatment failure. Additionally, even when alternate drug combinations can control mutated strains,
patients must rework their entire treatment strategy, become accustomed to new and more severe side effects,
and deal with a high viral load until the new treatment becomes effective [3]. When the virus doesn’t mutate,
noncompliance still means an increased viral load, vastly increasing transmission rates.
These dangers have exacerbated the stigma around HIV [4], leading to further noncompliance [6]. It is a
vicious cycle to which young people are particularly susceptible [4]. Since the virus can mutate even without
noncompliance, young people in developing countries are at very high risk of treatment failure.
To help relieve the burdens and risks inherent in daily oral administration of ARVs, James J. Hobson, Amer Al-
khouja, et al. used a prodrug formulation to create semi-solid nanoparticles containing the nucleoside reverse
transcriptase inhibitor emtricitabine (which is commonly used to treat HIV) (cite paper here). Their analysis
predicts sustained prodrug release, so this isand represents a step toward injectable, long- acting HIV
treatment.

Methods

The researchers synthesized sixteen separate prodrugs, by attaching carbonate and carbamate groups. To
synthesize 5’-alkoxycarbonyl emtricitabine (FTC) carbamates (1–8), they suspended emtricitabine in
dichloromethane. Then, they added 3 eq. of 1.5M pyridine and cooled the mixture to 0 oC in an ice water bath.
After cooling, they initiated the reaction with dropwise addition of alkyl chloroformate. The reaction was
monitored with thin layer chromatography (TLC) and completed in three hours. Upon the reaction’s completion,
they removed volatiles under reduced pressure. To purify the resulting product, they used silica flash
chromatography.
Synthesizing emtricitabine carbamates 9-16 began with dissolving 1 eq. of .5M 5’-alkoxycarbonyl FTC
carbamates (1-8) in tetrahydrofuran. The researchers added 5 eq. of 2.5M lithium hydroxide, a strong base,
and approximately 20 drops of water to improve the mixture’s solubility. Then, monitoring the mixture with TLC,
they stirred it until the reaction was complete. As they did for prodrugs 1-8, the researchers removed volatiles
under low pressure, then purified the product with silica flash chromatography. Both methods created prodrugs
with range of hydrophobic alkyl carbonate and/or carbamate groups, resulting in less water-soluble particles to
process using emulsion-templated freeze drying.
To generate the carbonate cleavage initial rate measurements, they preincubated reaction mixtures containing
phosphate buffer and mixed gender human skeletal muscle or mixed gender human liver at human body
temperature (37oC) for five minutes. To initiate the reaction, they added 1 mM prodrugs + 5% dimethyl
sulfoxide. Then, maintaining a constant temperature, they took aliquots measuring initial rate periodically.
Intervals depended on the prodrug and solution, but every combination included an initial measurement at half
a minute, and all measurements were completed within 5 hours. Each aliquot was submerged in ice cold
methanol, then centrifuged for five minutes. The researchers diluted the supernatant with nine times its volume
of phosphate buffer. Then, they used high performance liquid chromatography (HPLC) to analyze the mixture.
To determine how long the prodrugs would survive in human plasma, the researchers preincubated mixed
gender human plasma at 37oC, then added the prodrugs to initiate the reaction. Maintaining the temperature,
the authors took periodic samples, quenched them in cold methanol, then centrifuged them. The resulting
supernatant was diluted with a phosphate buffer with the pH of plasma, then and analyzed with HPLC.
To provide guidance for options containing higher drug concentrations, the researchers performed accelerated
screening with particles with 10 weight % prodrug loading. This emulsion-templated freeze-drying synthesis is
a useful way to establish robustness and repeatability with fewer repeats. To synthesize, the authors fully
dissolved polymers and surfactants for the nanoparticle coating in water at a concentration of 22.4 mg/mL.
. Each surfactant and polymer were chosen for its stabilizing effects, biocompatibility, and low toxicity. For
example, PEG is frequently used to reduce the immune response nanoparticles face while in circulation, and
Tween 20 is a common pharmaceutical excipient [5]. The researchers studied a total of 42 polymer/surfactant
combinations. This is significant; having so many combinations to stabilize and decrease immune response is
important because many people developing a resistance to PEG, so it is good to have alternate options [6]. To
create the semi solid nanoparticles, each prodrug was dissolved in methanol then combined with every one of
the combinations. The samples were sonicated at 4oC, then frozen, freeze dried, and frozen againsubjected to
freeze-thaw cycles, and . After 48 hours, the researchers analyzed them at room temperature using dynamic
light scattering (DLS).
With the results of their analyses, the authors performed in vitro to in vivo extrapolation. Using the results as
parameters, they used mathematical modeling to allow them to make predictions about how their nanoparticle
would perform in a live bodyvivo [7].

Results

ARV drugs function in the blood plasma as that is where the HIV virus resides [8]. So, prodrugTherefore,
prodrug nanoparticles must be designed in a way that lets them activateactivates the drug by the time theytime
the particles reach the bloodstream to have maximum effect before they are cleared out. To achieve this, the
authors modified the FTC prodrug in two ways: by attaching varied chain lengths of alkyl chloroformate to both
the amine and hydroxyl functional group, or by adding the alkyl chloroformate only to the amine functional
group. They successfully synthesized eight different chain lengths per method for a total of 16 prodrug
analogues. Each of these analogues has a different hydrophobicity and physical property. After successfully
synthesizing 16 different prodrug analogues, they performed HPLC to see the activation of the FTC prodrug in
different biologically relevant conditions. The molecules that were modified at the hydroxyl and amine groups
had a much shorter half-life (<12h) for all chain lengths than those that had a modification only at the amine
group (prodrugs 9-16). Prodrugs 1-8 had a longer half-life (>12h), with a downward trend of half-life with
increasing chain length, ranging up to >50h with the shortest chain length, revealed much greater stability in
blood plasma. While in the plasma, prodrug stability increased with longer chain length for prodrugs 1-8, in the
presence of muscle and liver subcellular fractions, hydrolysis rates reached a maximum with the 3-carbon
chain length, with a decrease in hydrolysis for longer and shorter chain lengths. For all chain lengths, the
hydrolysis rate was an order of magnitude larger in the liver than it was in the muscle. Prodrugs with a single
modification at the amine group showed a virtually nonexistent hydrolysis rate.
After characterizing the synthesis and biological activation of FTC, the researchers performed a series of
studies to determine the best polymer and surfactant to coat their semi-solid prodrug nanoparticles (SSPNs).
First, they performed an accelerated screening approach, using DLS to define the re-dispersion, z-average
diameter, stability, and uniformity of the SSPNs. Seven polymers, six surfactants and a 10% drug loading
yielded 42 candidate SSPNs per prodrug analogue, leading to a total of 672 total possibilities. These
possibilities were ranked against the 16 prodrug analogues. The molecules with the modification only at the
amine group and the 1 to 3-carbon chain length molecules with both modifications were compatible with less
than five combinations each. In the molecules with both modifications and those with more than 4-carbon chain
lengths, more than half of the potential candidates produced hits. There was a discrepancy while comparing
the hydrophobicity to the ability to form the SSPNs, as there was a difference between compounds with similar
hydrophobicity and the number of hits.
The initial studies were done using a low drug loading percentage. Since this percentage is insufficient for a
depot injection, they performed the same series of studies with a higher drug loading of 50% and 70%. This
time they only utilized compounds with both modifications and carbon chain lengths of 4, 6, and 8. As the drug
loading increased, there were fewer hits. For the 50% drug loading, there were 36 hits, and for the 70% drug
loading, there were only 4 hits. This showcased the difficulty of predicting a nanoparticle formulation that meets
all criteria necessary for the human system. The formulation remained stable across a period of several
months and transatlantic transportation.
Due to the complexity of this system and the difficulty inherent in developing a nanotherapeutic that meets all
design criteria, they used computational methods to predict in vivo pharmacokinetics. This is a way to ensure
that future in vivo studies are well designed to minimize the number of animals needed. They first compared
the in vitro activation of FTC and compared it to the human pharmacokinetics (PK) of FTC. The total release, o,
measured as ther the area under the curve, varied by +76%, while the maximum and minimum points varied
by +62% and -10%. Then, they proceeded to model FTC with both modifications and either 4 or 8-carbon
chain length over 28 days, showing the released to the FTC with only the amine modification, and finally the
original FTC molecule. FTC with one modification showed very rapid hydrolysis, which had a very small
contribution to the total plasma concentration.

Weaknesses and Strengths

As the researchers mention in their paper, when designing a nanoparticle platform, there are many competing
biological processes to consider after administration. They were able to take into account the biological
activation of the drug upon entering the body by successfully chemically modifying it and learning the
hydrolysis that occurs. They also explored all different options when it came down to choosing a polymer and
surfactant to use for their studies. While they investigate the effect of FTC activation in biologically relevant
conditions by exposing them to blood plasma, and subcellular fragments of liver and muscle, they account for
all components present in the body that can affect biodistribution. While developing the best SSPN, they did
not consider engineering the surface to improve circulation and targeting of the drug, or how the polymer and
surfactant would affect the body. This could have been addressed with a toxicity study, a widely used
preliminary study using cells [9]. While they did not perform any in vivo studies, which would be the best way to
measure the PK of the drug, they did perform modelling studies to gather more information for future in vivo
studies, which was not part of this specific project.  Additionally, there were contradicting factors that could
have been better included when deciding which formulation to use for further studies. As an example, they
found that the 3-carbon analogue had the highest hydrolysis rate but decided to not further investigate the
compound. They explained it as if they only took into account the number of hits they got for that compound as
a measure to discard it for future studies. When they performed the modelling studies, they chose a 70wt%
drug loading, while they were only able to achieve that formulation for a few combinations of polymer and
surfactant.

Potential Implications

This paper showcased the value of using in vitro to in vivo modeling to quickly determine which nanoparticles
have the most potential for sustained drug release and reactivity to appropriate drug environments. Though
accounting physiology was not an active priority, the wide scope of the study is an excellent springboard for
future work into long acting, injectable HIV treatment. The use of semi-solid nanoparticles outside of topical
drug applications is also relatively new. This study could prompt further research into the potential of semi solid
nanoparticles for drug delivery.  
The enormous number of polymer and surfactant combinations is useful for future studies; each of these has
stabilizing and immune response decreasing capabilities. These combinations offer an alternative to
researchers and drug developers who can no longer shield their products with PEG due to increased immune
response.
This paper is a start to relieving the monotony of daily oral ingestion of HIV medication. Continuing the
research could turn HIV into a more manageable infection, reduce transmission rates, and decrease instances
of drug mutation. HIV positive people would enjoy a higher quality of life and could face less stigma as the
burden of treatment and risk of transmission decreases.
References

1. Heckman, B. W., Mathew, A. R. & Carpenter, M. J. Treatment burden and treatment fatigue as barriers to
health. Curr. Opin. Psychol. 5, 31–36 (2015).
2. "HIV/AIDS Fact sheet N°360". WHO. November 2015. Archived from the original on February 17, 2016.
3. "HIV Classification: CDC and WHO Staging Systems | AIDS Education and Training Centers National
Coordinating Resource Center (AETC NCRC)"AIDS Education and Training Center Program. Archived from
the original on October 18, 2017.
4. Herek GM, Capitanio JP (1999). "AIDS Stigma and sexual prejudice" (PDF). American Behavioral Scientist.
42(7): 1130–47. doi:10.1177/0002764299042007006. Archived
5. Ayorinde FO; Gelain SV; Johnson JH Jr; Wan LW. (2000). "Analysis of some commercial polysorbate
formulations using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry". Rapid
Communications in Mass Spectrometry. 14 (22): 2116–2124. doi:10.1002/1097-
0231(20001130)14:22<2116::AID-RCM142>3.0.CO;2-1
6. McSweeney, MD.; et al. (2018). "Physician Awareness of Immune Responses to Polyethylene Glycol‐Drug
Conjugates". Clinical and Translational Science. 11 (2): 162–165. doi:10.1111/cts.12537
7. Quignot, Nadia; Bois, Frédéric Yves (2013). "A computational model to predict rat ovarian steroid secretion
from in vitro experiments with endocrine disruptors". PLoS ONE. 8 (1): e53891.
doi:10.1371/journal.pone.0053891
8. Arts JE, Hazuda DJ. HIV-1 Antiretroviral Therapy. Cold Spring Harb Perspect Med. 2012 Apr; 2(4):
a007161.
9. Allen DD, Caviedes R, Cárdenas AM, Shimahara T, Segura-Aguilar J, Caviedes PA. Cell lines as in vitro
models for drug screening and toxicity studies. Drug Dev Ind Pharm. 2008 Feb;34(2):234.