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Long-Acting, Potent Delivery of Combination Antiretroviral Therapy. Anna H. F. ... Publication Date (Web): April 30, 2018. Copyright © 2018 American ...
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Letter Cite This: ACS Macro Lett. 2018, 7, 587−591

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Long-Acting, Potent Delivery of Combination Antiretroviral Therapy Anna H. F. Andersen,†,‡ Camilla F. Riber,† Kaja Zuwala,†,‡ Martin Tolstrup,‡ Frederik Dagnæs-Hansen,§ Paul W. Denton,‡ and Alexander N. Zelikin*,†,∥ †

Department of Chemistry, Aarhus University, Aarhus 8000, Denmark Department of Infectious Diseases and Department of Clinical Medicine, Aarhus University Hospital, Aarhus 8200, Denmark § Department of Biomedicine, Aarhus University, Aarhus 8000, Denmark ∥ iNano Interdisciplinary Nanoscience Centre, Aarhus University, Aarhus 8000, Denmark ‡

S Supporting Information *

ABSTRACT: Antiretroviral therapy (ART) has revolutionized HIV treatment, yet grand challenges remain: (i) short blood and body residence time of the antiviral drugs, (ii) relative poor antiretroviral drug penetrance into key tissue reservoirs of viral infection, namely, the spleen and lymph nodes, and (iii) obstacles in different pharmacokinetics of the necessary combination drugs. We present a novel drug delivery approach that simultaneously overcomes these limitations. We designed albumin−polymer−drug conjugates where albumin ensures long body residence time as well as lymphatic accumulation of the conjugate. The polymer enabled the delivery of combinations of drugs in precise ratios affording potency superior to the individual antiretroviral drugs and strong protection from HIV infection in primary human T cells.

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each cell precisely in the nominated ratio. To realize these opportunities, in this work we (i) designed the synthesis of APD via orthogonal conjugation of the polymer with the antiretroviral drugs and the protein; (ii) selected the lead polymer−drug composition to achieve potent, efficacious delivery of ART; (iii) characterized the in vivo biodistribution of APD; and (iv) validated the potent antiretroviral activity of APD in model cell culture and in primary human CD4+ T cells. First, we aimed to verify if conjugation of macromolecules to albumin confers to the synthetic polymer the receptor recognition and the pharmacokinetic attributes of albumin. The polymer was N-(2-hydroxypropyl) methacrylamide (PHPMA)due to the previous successes of PHPMA as a backbone for macromolecular prodrugs (MP).17,18 Polymer synthesis was conducted using the reversible addition− fragmentation chain transfer polymerization technique (RAFT).19 We used the 2-cyano-5-oxo-5-(2-thioxothiazolidin3-yl)pentan-2-yl dodecyl carbonotrithioate chain transfer agent (CTA). Upon polymerization, 2-thioxothiazolidin-3-yl comprises the polymer end group for one-step conjugation with protein amine groups. This was used to link the polymer chains to albumin. We used a sample of PHPMA with a molar mass of 7 kDa, which is well within the renal secretion limits (ca. 30 kDa). Conjugation to albumin was performed to install ca. 1 polymer chain per protein globule. Affinity of the PHPMA− albumin conjugate to the human FcRn receptor (responsible for albumin recycling and long body and blood residence time12,13) was similar to that of pristine albumin (Figure 1A)

ntiretroviral therapy (ART) for treating human immunodeficiency virus (HIV) infection is arguably one of the best success stories of medicinal chemistry.1−3 The cornerstone of current ART is the use of combinations of antiretroviral drugs to effectively suppress the virus and prevent drug resistance development.4,5 However, a key challenge remains in that antiviral drugs exhibit a short plasma half-life which necessitates frequent drug administration. To address this, continuous efforts are being made to develop long-acting ARTdelivery formulations.6,7 Another challenge is that, despite therapy, the virus remains in the body in a transcriptionally active form in lymphoid tissues8,9 likely due to poor penetration of ART into these tissues.8−11 From these sanctuary tissues and from the latent state within the CD4+ T cells, HIV can rebound and quickly establish viremia equal to pretreatment levels in patients not adhering to the stringent regimen of ART. We considered whether key challenges for HIV maintenance can be addressed using a product of nature, namely, albumin. Albumin is the most abundant protein in human plasma, is inert, and is nonantigenica key safety feature. This protein has a phenomenal blood residence time (19 days in humans, 24 h in mice)12,13 which serves as a highly successful tool to create long-acting formulations for the associated therapeutic cargo.14,15 Also, albumin has a natural propensity to localize in the lymph nodes and spleen.16 We develop albumin−polymer−drug conjugates (APD) such that albumin is responsible for pharmacokinetics of the formulation, whereas the polymer arm allows that multiple dissimilar drug molecules comprising ART are conjugated to the same carrier polymer. In such formulations, all drugs have the same pharmacokinetics; the drug−drug ratio is strictly set by the polymer composition; and the drugs are delivered to © XXXX American Chemical Society

Received: March 5, 2018 Accepted: April 25, 2018

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DOI: 10.1021/acsmacrolett.8b00179 ACS Macro Lett. 2018, 7, 587−591

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ACS Macro Letters

of APD from blood is compensated by translocation of APD from the subcutaneous depot. Even at the 24 h time point, concentration of APD in blood remained on a high level of ca. 30% and at 48 h on a ca. 20% level of the peak concentration. This body residence time well exceeded that of PHPMA not conjugated to albumin (for the latter: 2 h half-life for i.v. administration, constant blood level following the onset for only 2 h). Localization of the injected payload was visualized on an in vivo imaging system (IVIS) (Figure 1C, for brevity only results of i.v. drug administration are shown). These images illustrate that within 60 min after i.v. administration PHPMA is localized in the bladder, and over 24 h the entire payload of the administered polymer was eliminated from mice. In contrast, the albumin-conjugated polymer did not reveal renal excretion over a short time frame, and after 24 h mice exhibited minor changes in the levels of fluorescence. Together, results in Figure 1B,C validate the extended blood and body residence time of PHPMA when conjugated to albumin. Sacrificed mice were used to visualize and quantify fluorescence of the polymer/APD in the harvested organs (Figure 1D,E). Fluorescence images demonstrate that APD exhibits the highly desired accumulation in lymph nodes. This was apparent for i.v. administration during observation between 1 and 24 h (Figure 1D) and on a longer scale, for s.c. administration, when comparing organs harvested at 1 day and 7 days. Visual observations are well supported by quantitative measurements of fluorescence (Figure 1E). While PHPMA itself exhibited a noticeable trend of lymph node localization, this behavior was markedly enhanced by conjugating the polymer to albumin. In the latter case, the payload progressively, significantly accumulated in lymph nodes over a 7 day observation time (APD, S.C. p = 0.047), whereas in other organs payload peaked at day 1 and decreased to day 7. Taken together, results in Figure 1 provide strong support for the proposed approach of using APD as a long-acting antiviral delivery strategy also poised to achieve delivery to tissue reservoirs of HIV infection and specifically lymph nodes. The advantageous biodistribution of the APD encouraged us to develop MPs as carriers for a combination of antiretroviral drugs. This was achieved through copolymerization of monomers with functionality of azidothymidine (AZT)21 and lamivudine (3TC).22 The monomers were designed such as to contain a disulfide linkage paired to a self-immolative liker (SIL)for efficient intracellular drug release23−26 (Figure 2). For polymers carrying a combination of AZT and 3TC, similarity in monomer structure afforded polymers with composition closely matched to the monomer feed ratio (see Table 1 and Supporting Information for full details). Polymers were purified through precipitation and analyzed for molar mass using size exclusion chromatography and per monomer composition using NMR (Table 1 and Figure S1). Antiretroviral effects elicited by the polymers were quantified using HIV-1Bal27 and the TZM-bl cells.28 Dose response curves obtained for each polymer−drug conjugate (Figure S2) were used to derive the IC50 values for all MP and APD, in Table 1. We tested combinations of AZT and 3TC in three different ways: (1) MPs for AZT and for 3TC taken individually, (2) mixtures of the prodrugs to test coformulation of the drugs, and (3) MPs whereby each polymer chain concurrently carries both AZT and 3TC, affording a true coadministration and codelivery of drugs on the cellular level. IC50 values for MP as expressed in equivalent concentration of the conjugated drug were margin-

highly important with the view of creating APD with biodistribution properties of albumin.

Figure 1. (A) Affinity of albumin and the albumin−polymer conjugate to the FcRn receptor. (B) Concentration of the albumin−polymer conjugate and the parent polymer in blood upon intravenous or subcutaneous administration quantified over a period of 7 days using fluorescently labeled samples and fluorescence measurements on drawn blood samples performed on IVIS. Each data point represents an average of 4 mice ± SD. (C) Full body and (D) representative organ fluorescent images obtained on IVIS. For (C) and (D) fluorescence on images can be directly compared longitudinal between identical treatments. LN: lymph nodes. (E) Quantification of fluorescence in harvested organs for PHPMA and APD conjugates following i.v. and s.c. drug administration, presented by the fold change compared to the 1 h time point. Each data point represents an average of 4 mice ± SD (lines are guides to the eye only and do not reflect longitudinal study). Fold changes compared to 1 h were logtransformed and analyzed by using a 1-way ANOVA with Bonferroni’s multiple comparisons: * p < 0.05; *** p < 0.001. Full statistical evaluation of data shown in panel E is listed in the Supporting Information Table S2.

For in vivo studies, PHPMA and APD were labeled with Cy7 fluorophore through a nondegradable linker, and samples were administered to mice through intravenous (tail vein) or subcutaneous injection (scruff of the neck). Periodic sampling of blood was performed to quantify the plasma polymer or albumin−polymer conjugate levels. Intravenous administration of APD resulted in a typical concentration decay profile with a half-life of ∼3.4 h. There was no plateau phase of APD residence in peripheral blood following intravenous administration of APD (Figure 1B). In contrast, subcutaneous administration (as is employed for administration of the albumin-bound drugs, e.g., Liraglutide20) afforded a highly desirable pharmacokinetic profile whereby the concentration of APD in blood remained on a constant level for ∼9 h, following a 2 h onset period. During this time, it is likely that elimination 588

DOI: 10.1021/acsmacrolett.8b00179 ACS Macro Lett. 2018, 7, 587−591

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superior to PHPMA-AZT, and superior to the combination therapy based on the mixture of MPs conjugated to individual drugs (Table 1). For the polymer composed of a 6:4 ratio of AZT and 3TC, IC50 value was as low as 14 nM expressed in the equivalent concentration of the two drugs combined. This result is highly important because it illustrates the benefits of MP-based combination ART. These data also highlight that the ratio of the two drugs is critically important in MP efficacy. Specifically, we found that the optimized (6:4 ratio) formulation exhibited potency well exceeding that of the pristine drugs. Finally, to produce the desired APD, the PHPMA-AZT-3TC polymer was conjugated to albumin. The majority of albumin− polymer conjugates described in the literature undergoes selfassembly into nanoparticles,30−32 and while these tools of nanotechnology may be powerful in their own right, there is limited clinical evidence to suggest that pharmacokinetics of these aggregated proteins is beneficial for drug delivery. In contrast, molecularly dissolved albumin exhibits extended blood and body residence time and translocates to lymphoid tissues and has been used in clinical practice for drug delivery.33,34 It is highly important that APD synthesized in this work remained colloidally stable, as illustrated by the SEC profiles (Supporting Information Figure S3.) APD had a molar mass of 81.8 kDa, reflecting a molar mass increase of 14 kDa per albumin globule, that is, ca. 1 polymer chain attached per albumin. The dispersity for APD was 1.093 (1.015 for pristine albumin), revealing a uniform distribution of polymer chains between protein globules. Each APD contained on average 10 drug molecules per conjugate (4 copies of 3TC and 6 copies of AZT), that is, 10-fold higher drug loading than commercially successful albumin-based drug delivery products (Liraglutide, Semaglutide). The resulting APD excelled from the perspective of both potency and efficacy of delivery of ART and effectively suppressed infectivity of HIV with IC50 values ∼0.3 mg/L total conjugate or 21 nM expressed through the concentration of the conjugated drugs (Figure 3A).

Figure 2. (A) Chemical formulas of HPMA (1), RAFT agent (2), and AZT- and (3) 3TC-containing monomer (4) used for the synthesis of MP. (B) Schematic illustration of the synthesis of MP through copolymerization of HPMA and the monomers containing AZT and 3TC. (C) Illustration of the synthesis of the albumin−polymer−drug conjugate through covalent conjugation of MP to albumin.

Table 1. Macromolecular Characteristics of the MP and APD and Activity-Related IC50 Values for AZT, 3TC, MP, and APD Determined in TZM-bl Cells and Infected with HIV1Bal Virus Mn; Đ; drug content AZT 3TC PHPMA-AZT PHPMA-3TC PHPMA-AZT + PHPMA-3TC PHPMA (AZT + 3TC)

APD

AZT:3TC

IC50, nM

4:5 3:7 6:4 4:5 3:7 6:4 6:4

75 403 147 445 110 50 27 115 25 14 21

7.8 kDa; Đ 1.2; 16% 7.5 kDa; Đ 1.1; 7%

18.4 kDa; Đ 1.1; 10% 20.3 kDa; Đ 1.1; 10% 15.9 kDa; Đ 1.1; 9% 81.8 kDa; Đ 1.1

Figure 3. (A) Dose response curve for inhibition of infectivity of HIV1BaL strain in TZM-bl cells using an APD that contained an average of 6 copies of AZT and 4 copies of 3TC conjugated to the same carrier PHPMA chain. N = 3 ± SEM. (B) Time course of HIVHxB2D infection primary T cells from human donors upon treatment with a combination of AZT and 3TC formulated using pristine drugs, their MP, or APD. N = 2 ± SEM.

ally but noticeably higher than that for the corresponding pristine drugs, as is standard and well documented for MP.29 Coformulation of AZT and 3TC using mixtures of individual MPs was performed at or around a 2:1 mass ratio of AZT to 3TC (similar to the AZT to 3TC ratio in the approved combination therapy marketed as Combivir). Combination treatments exhibited potency that was significantly superior to either PHPMA-AZT or PHPMA-3TC individually (Figure S2 and Table 1). Furthermore, MPs based on PHPMA containing both AZT and 3TC were superior to the pristine drugs,

To directly demonstrate the efficacy of APD in a physiologically relevant setting, we used primary CD4+ cells harvested from healthy donors and set up a viral replication assay where cells were infected in the presence of antiretroviral drugs, MP, or APD (in each case, 1 μM AZT + 0.78 μM 3TC). During the course of 4 days, an uninhibited virus spread in the 589

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cells with an almost thousand-fold increase in levels of the viral p24 (Figure 3B). With ART treatment, replication was limited to a plateau at ca. 1000 pg/mL of p24, a concentration that stabilized at 48 h after infection. MP-incubated cells exhibited inhibited viral spreading as p24 levels in these cultures plateaued at approximately 4500 pg/mL. Most importantly, with APD treatment, virus spreading was effectively inhibited, and p24 concentration in the cultures did not exceed 2000 pg/ mL. This highlights the robust antiviral efficacy of APD. Of note, no antiviral activity was observed in the cells incubated with albumin (without conjugated polymer or drugs). These data demonstrate that long-acting APD formulations carrying an optimized number of antiretroviral drug molecules are able to efficiently reduce (∼50-fold) the spread of HIV in primary human T cells. Albumin−polymer−drug conjugates deliver potent combinations of antiretroviral drugs with a highly favorable biodistribution profile. These APDs capitalize on the natural propensity of albumin to exhibit extended body and blood residence time and translocate into lymphoid tissues phenomena highly desired but missing in conventional drugs currently comprising ART. We developed APDs that contained approximately 10 drug molecules per conjugate, exhibited inhibition of HIV at IC50 of 21 nM, and inhibited the spreading of HIV in primary human T cell cultures. Compared to recent reports on this subject,7,35 our method for ART delivery appears to be superior in that it formulates combination therapy for delivery in a strict ratio to each individual cell and facilitates lymphatic drug delivery, the two aspects pivotal for maintenance of HIV. We are now developing APD using retroviral drugs that represent the current benchmark in the treatment of HIV.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.8b00179. Syntheses and characterization of monomers and polymers, bioconjugation procedures, protocols for cell culture and animal experiments, dose response curves for inhibitions of viral proliferation, microscopy visualization of polymer, and APD cell entry (PDF)

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Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Anna H. F. Andersen: 0000-0003-0681-3611 Alexander N. Zelikin: 0000-0002-9864-321X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Kaja Borup Løvschall for assistance with SEC analyses. We wish to acknowledge financial support from the Danish Council for Independent Research, Technology and Production Sciences, Denmark (ANZ; project DFF − 4184-00177) and from amfAR, The Foundation for AIDS Research (grant No 109328-59-RGRL). 590

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DOI: 10.1021/acsmacrolett.8b00179 ACS Macro Lett. 2018, 7, 587−591