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Abstract

Tenofovir alafenamide fumarate is a lipophilic prodrug of tenofovir which is preferentially metabolized in lymphatic tissue resulting in high concentrations of tenofovir (TFV) and its active diphosphate metabolite inside the cells that replicate HIV. Due to its selectivity for these tissues, lower total doses of TAF can be administered relative to tenofovir disoproxil fumarate (TDF) which results in improved bone and renal biomarkers. Tenofovir alafenamide fumarate has become the “backbone” of multiple combination products for the treatment of HIV, combined with emtricitabine for PreP and as a monotherapy for the treatment or HBV.

Keywords

tenofovir tenofovir alafenamide fumarate HIV HBV PrEP nucleotide nucleoside

You must innovate your own products, if you don’t, someone else will.

John C. Martin

Discovery of TAF

Tenofovir (TFV) belongs to a class of acyclic nucleotide analogs which mimic the natural monophosphate nucleotides (Figure 1) [1].

Figure 1.

Structures of tenofovir (TFV), tenofovir DF (TDF), and TAF [2]

Tenofovir is readily phosphorylated inside cells to the diphosphate (TFV-DP), an analog of 2′- deoxyadenosine-triphosphate, by cellular kinases. TFV-DP is a very poor substrate and inhibitor for host DNA polymerases, but is a potent inhibitor of both HIV and HBV reverse transcriptases [3,4]. The structural simplicity of TFV retards resistance selection and results in favorable resistance profiles against nucleoside-resistant mutants. Inside peripheral blood mononuclear cells (PBMC) from patients, TFV-DP has an intracellular half-life of 150 h [5]. These intracellular pharmacological properties are ideal for treatment of HIV and HBV; however, TFV is a dianion at physiologic pH and poorly permeable to cell membranes, resulting in low cellular permeability and poor oral bioavailability.

To realize the potential of TFV in chronic antiviral therapies, it was necessary to have a product that could be delivered by the oral route of administration. In the mid 1990s, we initiated a TFV prodrug program to increase the absorption across the intestinal membrane and deliver TFV to the systemic circulation. These efforts led to the discovery of the tenofovir disoproxil fumarate disoproxil (TDF), a lipophilic prodrug of TFV and its subsequent clinical development in HIV [6,7]. In 2001, the FDA approved TDF (Viread) at a 300-mg dose for the treatment of HIV in combination with two other antiretroviral drugs. Subsequently, TDF was approved around the world and became the “backbone” of choice for the treatment of HIV. The rates of discontinuation due to toxicity or viral resistance are extremely low with TDF; however, its use has been associated with decreased renal function and bone mineral density and is restricted to patients with normal renal function (GFR >60 ml/min) [8,9].

Although TDF was designed to be rapidly metabolized in blood, in preclinical dog studies, the intracellular concentration of TFV in PBMCs was 5-fold higher after oral administration of TDF compared to an equivalent subcutaneous exposure of TFV [10]. In human clinical studies, the viral load drop after oral administration of TDF was 3-fold greater than with a similar exposure after IV administration of TFV [11]. These results suggested that TDF was doing more than just delivering TFV systemically, it was “enhancing” the intracellular distribution of TFV, likely due to the exposure of the gut lymphatic tissue to the intact prodrug prior to metabolism in the blood. This realization led us to ask the question, can we make a next generation prodrug which will have even greater distribution to tissues outside the gut, thus increasing intracellular concentrations in cells throughout the body. Critical to this approach was to stabilize the prodrug to plasma esterases while maintaining intracellular instability. Our initial strategy was to filter our selection of prodrugs using a series of in vitro assays designed to look at cellular HIV activity, MT-2 cell extract stability, and human plasma stability. The ideal prodrug would have increased potency and increased human plasma stability relative to TDF. Once identified, prodrugs would be optimized for oral bioavailability and intracellular loading of PBMCs.

Using the general approach described above, a variety of prodrug moieties from our previous work with adefovir and TFV were screened [12]. Most promising was a class of amino acid ester prodrugs previously explored by Chris McGuigan for AZT and d4T. These nucleoside phospho-amidates were designed to overcome the initial phosphorylation of nucleoside analogs which can be rate limiting in the formation of the nucleoside triphosphate [13,14]. In the case of TFV, the acyclic phosphonate itself is a monophosphate analog and the amidate esters function to mask the dianion, increase permeability, and change the cellular distribution of TFV. Extensive screening of this class of prodrugs led us to select a mono phenyl, mono-(L)-isopropyl alanine ester (GS-7171) as our lead candidate [2]. The synthesis of GS-7171 was non-selective resulting in a mixture of diastereomers due to stereochemical centers at both the amino acid and the phosphorous. The in vitro screens used to characterize the individual isomers are shown in Table 1.

Table 1.

In vitro anti-HIV-1 activity (EC50), cytotoxicity (CC50), and in vitro metabolic stabilities of tenofovir and tenofovir prodrugs [2]

Produg	EC₅₀ (μM)	CC₅₀ (μM)	Selectivity index	t_1/2(min)
Produg	EC₅₀ (μM)	CC₅₀ (μM)	Selectivity index	MT-2 cell extract	Human plasma
Tenofovir	5.0 ± 2.6	6000 ± 2700	1250	NR	NR
Tenofovir DF	0.05 ± 0.03	50 ± 28	1000	70.1 ± 10.1	0.41 ± 0.20
GS 7171	0.01 ± 0.005	95 ± 37	9500	ND	ND
GS 7339	0.06 ± 0.04	>100	>1700	>1000	231 ± 72
GS 7340	0.005 ± 0.002	40 ± 29	8000	28.3 ± 7.4	90 ± 12
GS 7485 (^D-Ala)	10 ± 4	ND	ND	700	200
GS 7160	10 ± 2.5	ND	ND	ND	ND

As shown in the table, the in vitro activity of the amidates is greater than TDF which is a reflection of the greater stability of the amidates versus TDF in the cell media and their ready metabolism inside cells to TFV-DP. Both TDF and the amidates were lipophilic and relative permeabilities were not likely a factor in the activity assessment because of the 3 day exposure to drug in the media during the assay. The additional critical parameter in our selection was the stability in human plasma. As shown in Table 1, the amidates are >100x more stable than TDF. Interesting and suggestive of a stereoselective metabolism, the diastereomeric mixture of theD-Ala (GS-7485) was relatively inactive, similar to the very polar mono alanine metabolite (GS-7160).

To interpret this favorable in vitro data, we initiated a broad range of pharmacokinetic studies in dogs to examine the plasma and PBMC profile of the prodrugs and their metabolites after oral and IV administration. Distinct from TDF where we saw no circulating levels of intact prodrug, both diastereomers of GS-7171, GS-7340, and GS-7339 were observed in plasma after oral administration, confirming that we could get systemic exposure of the intact prodrug after oral administration [2]. The development of a diastereomeric mixture is fraught with manufacturing and regulatory issues. To select the optimal isomer, we compared the AUCs of TFV in PBMCs after oral dosing of the individual isomers (GS-7339 and GS-7340) in dogs. As shown in Figure 2, GS-7340, subsequently known as TAF, resulted in the highest intracellular AUCs as measured in PBMCs.

Figure 2.

Total TFV AUCs (0–24 h) in plasma and PBMCs after oral administration of TDF and TAF (GS-7340), GS-7339, and GS-7171 and subcutaneous TFV in dogs [2]

The relative AUCs of TFV in PBMCs in dogs for sc. TFV and oral TDF and TAF (1x, 5x and 140x) are also shown in Figure 2. Additionally, we carried out radiolabeled distribution studies on TAF which demonstrated an enhanced distribution (5.7–15x) to the iliac, axillary, inguinal, and mesenteric lymph nodes and the spleen relative to TDF. Importantly, we did not see an enhanced delivery to the kidney. The preferential loading of lymphatic tissue was startling and hopeful, especially considering that this is the target organ for HIV replication. These results were highly suggestive that the oral administration of a low dose of TAF could increase the intracellular concentration of TFV in lymphatic tissue and reduce exposure to the kidney. Not only was there a potential for greater potency due to the much higher TFV-PP levels in lymphatic tissue, but the reduced systemic levels of TFV could broaden the use of TFV in patients with reduced renal function.

Cellular and biochemical studies led to the isolation and identification of a lysosomal carboxypeptidase, cathepsin A, found in high concentrations in lymphoid tissue, as the critical enzyme necessary initiate the metabolism of TAF to TFV and eventually to TFV-PP [15]. The low permeability of the intermediate metabolites and the long intracellular half-life of TFV-PP lead to accumulation in lymphatic tissue. Studies in isolated CD4+T cells and macrophages, both reservoirs for HIV replication demonstrated that TAF is efficiently activated [16]. The intracellular activation mechanism is depicted in Figure 3.

Figure 3.

Mechanism of cell loading by TAF [17]

The hypothesis that TAF could be a safer and more effective prodrug for the delivery of TFV into HIV-infected cells was tested in a monotherapy study in HIV treatment-naïve patients. The study compared doses of 40 and 120 mg of TAF versus 300 mg of TDF following 14 days of monotherapy [18]. The mean HIV RNA changes at Day 14 were −1.57, −1.71, and −0.94, respectively. Concentrations of total TFV in PBMCs from these patients were consistent with a greater intracellular exposure in the TAF arms relative to TDF. With these data, TAF had clearly met the initial criteria for the “next generation” TFV prodrug. However, there were several major issues that needed to be resolved before a commitment to full clinical development. The first was safety, it was not known if the chronic administration of TAF, which had a significantly different distribution than TDF would result in new toxicities. This concern was highlighted during subsequent chronic studies where new signals were observed at high TAF doses. Secondly, there was no current process for the manufacture and isolation of TAF at a scale which would allow commercialization.

If the drug synthesis is too costly and the majority of patients won’t have access, we are not going to put it into development.

John C. Martin

TAF process development and manufacturing

The initial monotherapy clinical trial used TAF drug substance that was synthesized as a mixture of diastereomers (GS-7171) and then purified by a chromatographic technique known as simulated moving bed chromatography (SMBC). SMBC was only available at pilot scale during this time and only at a few sites around the world. In addition, the theoretical yield was limited to less than 50%. Commercial production using SMBC was conceivable although not at quantities and costs which would allow a global distribution of TAF. The solution to the large-scale production of TAF is beautifully described in a series of manufacturing patents [19,20].

Using a crystallization-induced dynamic resolution process in the presence of phenol and a base, the diastereomeric mixture could be enriched in TAF by precipitating out TAF while continuously epimerizing the drug substance remaining in solution. Greater than 90% of the GS-7171 could be converted to TAF. Another breakthrough achieved was the discovery of the hemi-fumarate salt. Tenofovir alafenamide can be crystallized as a free base; however, its physical and chemical properties are not optimal for a robust drug tablet formulation. By rigorous salt screening strategies, it was discovered that a 2:1 mixture of tenofovir alafenamide to fumarate yielded a salt that not only had superior stability but could also be purified as the single diastereomer from GS-7171. These manufacturing advances were critical in our decision to move forward with TAF, ensuring we had adequate drug supply for an aggressive clinical program and a way forward for commercial manufacturing. As a result of both process and lower dose, TAF is available for the majority of HIV patients in resource-limited countries at pennies per day.

The history of drug development is overdosing.

John C. Martin

TAF clinical studies

After completing chronic toxicity studies and developing a scalable manufacturing process, TAF clinical development was resumed. At this point, the goals were altered from the initial goal of greater potency. TDF had been in millions of patients as part of multiple HAART regimens which reduced viral loads to undetectable levels in most patients. The limitations of HIV therapy were not potency but safety and tolerability. In post-marketing analyses from the TDF Expanded Access Program, renal failure, tubular dysfunction and elevated serum creatinine were rare but significant (0.3%, 0.05%, and 0.1%, respectively) [21]. TDF-related nephrotoxicity risk factors include older age, underlying renal disease, and ritonavir-boosted regimens. These considerations led us to study lower doses of TAF with the goal of reducing overall systemic exposure of TFV while maintaining potency at least equivalent to TDF.

As a result of these considerations, we re-entered the clinic with a mono-therapy study in HIV treatment-naïve patients comparing 8, 25, and 40 mg of TAF to 300 mg of TDF during a 10-day study [22]. The results were striking, the 8 mg of TAF resulted in a viral load reduction similar to 300 mg TDF and the 25 mg dose showed a significantly greater drop than TDF (−1.46 versus −0.97 log10 copies/ml). The systemic exposure of TFV after 25 mg of TAF is approximately 1/10 that of 300 mg of TDF, proving that circulating TFV concentrations are unrelated to antiviral effect when comparing these prodrugs. This was further supported by measuring the intracellular concentrations of TFV in PBMCs where it was observed that the level of TFV was 7-fold higher in the 25 mg TAF-treated patients compared to 300 mg TDF. These data also confirm that PBMC concentrations of TFV are a good surrogate for lymphatic tissue since they represent only a small fraction of the lymph cells in the body that can replicate HIV. At the systemic exposure of TFV from 25 mg of TAF, the therapeutic index is greatly expanded relative to TDF 300 mg, and this dose was chosen for further clinical development.

The efficacy of TAF has been confirmed in multiple patient populations and with multiple combination partners [23–25]. It has been thoroughly studied in treatment-naïve adults and adolescents, treatment-experienced adults following regimen switches, and in renal impairment studies. The summary from all these studies is that there is no significant difference in virologic response as compared to TDF in any of these patient populations. However, there were significantly smaller increases in serum creatinine, less proteinuria and albuminuria, and smaller decreases in bone mineral density, all consistent with lower levels of circulating TFV.

Tenofovir alafenamide fumarate is currently approved in 5 fixed-dose combination products for the treatment of HIV: Descovy (TAF/emtricitabine), Genvoya (TAF/emtricitabine/elvitegravir/cobicistat); Odefsey (TAF/emtricitabine/rilpivirine); Biktarvy (TAF/emtricitabine/bictegravir); Symtuza (TAF/emtricitabine/darunavir/cobicistat). The lower mass of TAF versus TDF (25 versus 300 mg) allows for smaller STR tablet sizes, and in the case of Symtuza, the creation of an STR that was not otherwise possible. In combinations with a pharmacokinetic booster-like cobicistat or ritonavir, the dose of TAF is reduced to 10 mg, which results in the same plasma TAF exposure as the un-boosted 25 mg dose [26].

In 2019, the FDA approved the fixed-dose combination of emtricitabine/FTC (Descovy) for pre-exposure prophylaxis (PrEP) [27]. The approval was based on a global phase 3 trial that compared the safety and efficacy of Descovy to Truvada (emtricitabine/TDF) in 5300 adult cisgender men who have sex with men or transgender women who have sex with men.

Descovy achieved non-inferiority to Truvada and showed statistically significant advantages with respect to both renal and bone laboratory parameters.

The preferential loading of TAF in lymphatic tissue makes it an ideal drug for the treatment of HIV, but liver is also a tissue which is metabolically active and capable of efficiently metabolizing TAF to TFV. A 28-day monotherapy antiviral study in treatment-naïve HBV patients led to the selection of the 25-mg dose of TAF for further clinical development [28].

Two 96-week phase 3 studies in treatment-naïve HBeAg-positive or HBeAg-negative patients comparing 25 mg of TAF to 300 mg of TDF demonstrated similar viral load suppression and more favorable renal and bone laboratory parameters compared to TDF [29]. Interestingly, there was a faster rate of ALT normalization in these patients on TAF than TDF. A switch study from TDF to TAF confirmed the antiviral and safety results. Tenofovir alafenamide fumarate 25 mg (Vemlidy) was approved by the FDA in 2016 for the treatment of HBV [30].

Conclusion

The safety and efficacy of HIV treatments has continuously improved since the advent of HAART. TAF, a lymphatic targeting prodrug of TFV, has been a significant part of these improvements. TAF is now used globally as a “backbone” for multiple HIV combination therapies in single-tablet regimens and with emtricitabine for PrEP. The favorable antiviral properties of TFV, long intracellular half-life, potency, and resistance profile, coupled to a target-directed prodrug moiety make this possible.

Footnotes

Acknowledgments

I would like to thank Susan Edl of Gilead Sciences for her diligent and timely preparation of this manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

William A Lee

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