HIV protease
Darunavir (TMC114) acts on HIV-1 protease [1]
Darunavir (TMC114) targets HIV-1 protease, with an IC50 of approximately 0.003 μM and an IC90 of approximately 0.009 μM against laboratory HIV-1 strains and primary clinical isolates; for HIV-1(NL4-3) variants resistant to saquinavir, indinavir, nelfinavir, or ritonavir, the IC50 ranges from 0.003 to 0.029 μM, and for amprenavir-resistant variants, the IC50 is 0.22 μM [2]
Darunavir (DRV) exerts anti-HIV effects by inhibiting HIV replication in U1 macrophages[3]

Darunavir (TMC114, 1a) is similar to other protease inhibitors in terms of stability[1].
\nDarunavir (TMC114, UIC-94017) suppresses the infectivity and replication of all HIV-1_NL4-3 variants exposed to and selected for resistance to AG1341, Ro 31-8959, MK-639, or ABT 538 at concentrations as high as 5 μM (IC50s, 0.003 to 0.029 μM), though its effectiveness against variants of HIV-1_NL4-3 selected for resistance to VX-478 was lower (IC50, 0.22 μM)[2].\n

---

\nThe screening of known HIV-1 protease inhibitors against a panel of multi-drug-resistant viruses revealed the potent activity of TMC126 on drug-resistant mutants. In comparison to amprenavir, the improved affinity of TMC126 is largely the result of one extra hydrogen bond to the backbone of the protein in the P2 pocket. Modification of the substitution pattern on the phenylsulfonamide P2' substituent of TMC126 created an interesting SAR, with the close analogue TMC114 being found to have a similar antiviral activity against the mutant and the wild-type viruses. X-ray and thermodynamic studies on both wild-type and mutant enzymes showed an extremely high enthalpy driven affinity of TMC114 for HIV-1 protease. In vitro selection of mutants resistant to TMC114 starting from wild-type virus proved to be extremely difficult; this was not the case for other close analogues. Therefore, the extra H-bond to the backbone in the P2 pocket cannot be the only explanation for the interesting antiviral profile of TMC114. [1]

---

\n\nResearchers designed, synthesized, and identified UIC-94017 (TMC114), a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing a 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) and a sulfonamide isostere which is extremely potent against laboratory HIV-1 strains and primary clinical isolates (50% inhibitory concentration [IC50], ∼0.003 μM; IC90, ∼0.009 μM) with minimal cytotoxicity (50% cytotoxic concentration for CD4+ MT-2 cells, 74 μM). UIC-94017 blocked the infectivity and replication of each of HIV-1NL4-3 variants exposed to and selected for resistance to saquinavir, indinavir, nelfinavir, or ritonavir at concentrations up to 5 μM (IC50s, 0.003 to 0.029 μM), although it was less active against HIV-1NL4-3 variants selected for resistance to amprenavir (IC50, 0.22 μM). UIC-94017 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents. Structural analyses revealed that the close contact of UIC-94017 with the main chains of the protease active-site amino acids (Asp-29 and Asp-30) is important for its potency and wide spectrum of activity against multi-PI-resistant HIV-1 variants. [2]

---

\n\nAlthough antiretroviral therapy (ART) can suppress peripheral HIV, patients still suffer from neuroHIV due to insufficient levels of ART drugs in the brain. Hence, this study focuses on developing a poly lactic-co-glycolic acid (PLGA) nanoparticle-based ART drug delivery system for darunavir (DRV) using an intranasal route that can overcome the limitation of drug metabolic stability and blood-brain barrier (BBB) permeability. The physicochemical properties of PLGA-DRV were characterized. The results indicated that PLGA-DRV formulation inhibits HIV replication in U1 macrophages directly and in the presence of the BBB without inducing cytotoxicity. However, the PLGA-DRV did not inhibit HIV replication more than DRV alone. Notably, the total antioxidant capacity remained unchanged upon treatment with both DRV or PLGA-DRV in U1 cells. Compared to DRV alone, PLGA-DRV further decreased reactive oxygen species, suggesting a decrease in oxidative stress by the formulation. Oxidative stress is generally increased by HIV infection, leading to increased inflammation. Although the PLGA-DRV formulation did not further reduce the inflammatory response, the formulation did not provoke an inflammatory response in HIV-infected U1 macrophages. As expected, in vitro experiments showed higher DRV permeability by PLGA-DRV than DRV alone to U1 macrophages. [3]

---

Screening of known HIV-1 protease inhibitors against multi-drug-resistant viruses revealed potent activity of TMC126 against drug-resistant mutants; Darunavir (TMC114), an analogue of TMC126, showed similar antiviral activity against wild-type and mutant HIV-1 viruses. X-ray and thermodynamic studies demonstrated an extremely high enthalpy-driven affinity of Darunavir for both wild-type and mutant HIV-1 protease. In vitro selection of Darunavir-resistant mutants from wild-type virus was extremely difficult, unlike other analogues [1]
Darunavir (TMC114/UIC-94017), a nonpeptidic HIV-1 protease inhibitor containing bis-tetrahydrofuranylurethane and sulfonamide isostere, exhibited potent activity against laboratory HIV-1 strains and primary clinical isolates (IC50 ≈ 0.003 μM, IC90 ≈ 0.009 μM) with minimal cytotoxicity (CC50 for CD4+ MT-2 cells: 74 μM). It blocked the infectivity and replication of HIV-1(NL4-3) variants resistant to saquinavir, indinavir, nelfinavir, or ritonavir (IC50: 0.003–0.029 μM) at concentrations up to 5 μM, while showing lower activity against amprenavir-resistant variants (IC50: 0.22 μM). It also had potent activity against multi-PI-resistant clinical HIV-1 variants isolated from patients unresponsive to existing antiviral regimens. Structural analyses indicated that the close contact of Darunavir with the main chains of Asp-29 and Asp-30 in the protease active site contributed to its potency and broad-spectrum activity against multi-PI-resistant HIV-1 [2]
PLGA-nanoformulated Darunavir (PLGA-DRV) had an encapsulation efficiency of 84.19% ± 6.60%, drug loading of 1.94%, and particle size ranging from 111.1 nm to 175.1 nm. PLGA-DRV inhibited HIV replication in U1 macrophages both directly and in the presence of the blood-brain barrier (BBB) without inducing cytotoxicity (assessed by LDH activity), though it did not show stronger inhibition than free DRV. Treatment with PLGA-DRV or free DRV did not alter the total antioxidant capacity of U1 cells, but PLGA-DRV further reduced reactive oxygen species (ROS) levels, decreasing oxidative stress. PLGA-DRV did not exacerbate or induce an inflammatory response in HIV-infected U1 macrophages and showed higher permeability to U1 macrophages than free DRV [3]

Darunavir has a 37% oral bioavailability and is effective against both PI-resistant and wild-type HIV. In conjunction with ritonavir, it is frequently used to increase the bioavailability to 82%.

---

Pharmacokinetics. [1]
The second selection criterion was a set of pharmacokinetic related properties. In Table 6 the metabolic stability is presented in three different species (liver microsomes of rat, dog, and human origin) as % remaining parent compound after 30 min incubation at 37 °C. The degree of metabolism is determined by direct measurement of the residual parent compound in the reaction mixture using LC-MS. 1b and 1d appeared to be extremely labile. Darunavir (TMC114) had a stability comparable to other protease inhibitors. 2 and IDV have been included as a reference in Table 6.
The same trend was observed in oral absorption studies in animals. Data of oral administration to dogs at 80 mg/kg as a PEG400 solution are presented in Table 7. Darunavir (TMC114) is clearly superior to 1b and 1d both in terms of Cmax and AUC. During this evaluation, for compound 1b only minor levels of possible metabolite 1i were observed. In analogy with fosamprenavir, the monophosphate prodrug of 2, we studied the behavior of compound 1h, the monophosphate ester of Darunavir (TMC114). The main advantage of this type of prodrugs are their superior solid-state characteristics, which is outside the scope of this publication. We only investigated the potential for higher bioavailability. Data of a single oral administration in PEG400 in rats at 20 mg/kg were generated; the parent compound and the monophosphate behave in a similar way. For 2 and its prodrug, similar observations were reported previously.

---

Importantly, in vivo experiments, especially using intranasal administration of PLGA-DRV in wild-type mice, demonstrated a significant increase in the brain-to-plasma ratio of Darunavir (TMC114)/DRV compared to the free Darunavir (TMC114)/DRV. Overall, findings from this study attest to the potential of the PLGA-DRV nanoformulation in reducing HIV pathogenesis in macrophages and enhancing drug delivery to the brain, offering a promising avenue for treating HIV-related neurological disorders. [3]

---

Intranasal administration of PLGA-DRV nanoformulation in wild-type Balb/c mice at a dose of 2.5 mg/kg Darunavir resulted in a significantly higher brain-to-plasma ratio of DRV compared to free DRV. DRV concentrations in the brain, plasma, lungs, and livers of mice were measured at 1 h, 3 h, 6 h, and 12 h after intranasal (IN) or intravenous (IV) administration of PLGA-DRV or free DRV, confirming enhanced brain delivery of DRV by the nanoformulation [3]

Darunavir has a Ki of 1 nM for wild type HIV-1 protease.
\nIsothermal Titration Calorimetry. [1]
\nThermodynamic parameters of inhibitor binding were determined using an isothermal titration calorimeter, VP-ITC. The buffer used for all protease and inhibitor solutions consisted of 10 mM sodium acetate pH 5.0, 2% DMSO, and 2 mM tris(2-carboxyethyl)phosphine (TCEP). The binding affinities of 2 and Darunavir (TMC114) for the multi-drug-resistant protease were obtained by the displacement titration method, using acetyl-pepstatin and indinavir, respectively, as the weaker binder Direct titration experiments were also performed with the tightly binding inhibitor to confirm the enthalpy changes obtained by the displacement method. Each experiment was performed at least twice. The details of the ITC experiments have been published elsewhere.\n

---

\nGenotyping. [1]
\nGenotypic analysis was performed by automated population-based full-sequence analysis. Sequencing results are reported as amino acid changes compared to the wild-type (HXB2) reference sequence.\nInVitro Selection of Resistant Strains. MT-4-LTR-EGFP cells were infected at a multiplicity of infection of 0.01 to 0.001 CCID50/cell in the presence of the inhibitor compound at a starting concentration two to three times the EC50. The cultures were subcultivated and scored microscopically on virus-induced fluorescence and cytopathic effect every 3 to 4 days. Cultures were subcultivated in the presence of the same concentration of compound until full virus breakthrough, and subsequently at a higher compound concentration to select for variants able to grow in the presence of the highest possible inhibitor concentration.\n

---

\nX-ray Crystallography. [1]
\nA multi-drug-resistant HIV-1 protease with substitutions L63P, V82T, and I84V was crystallized in complex with Darunavir (TMC114) and 2. Both structures crystallized in the space group P212121 with one dimer per asymmetric unit. Data on the Darunavir (TMC114) complex were collected under cryocooled conditions using the synchrotron source at Advanced Light Source at Lawrence-Berkeley Laboratory, Berkeley, CA. The crystal complex with Darunavir (TMC114) diffracted to a resolution of 1.35 Å, with an R-factor of 16.8%. Data for the complex with 2 were collected at room temperature on an R-axis IV image plate system mounted on a Rigaku rotating anode source. The resolution obtained for the complex with 2 is 2.2 Å. The details of the X-ray crystallography experiments and the refinement statistics have been published elsewhere.15 The X-ray structures have been submitted to the Protein Data Bank (PDB-codes 1T7I and 1T7J).\n

---

\n\nDrug susceptibility assay. [2]
\nThe susceptibilities of HIV-1LAI, HIV-1Ba-L, HIV-2EHO, HIV-2ROD, and the primary HIV-1 isolates to various drugs were determined as described previously, with minor modifications. Briefly, MT-2 cells (2 × 104/ml) were exposed to 100 50% tissue culture infectious doses (TCID50s) of HIV-1LAI, HIV-1Ba-L, HIV-2EHO, or HIV-2ROD in the presence or absence of various concentrations of drugs in 96-well microculture plates and were incubated at 37°C for 7 days. After 100 μl of the medium was removed from each well, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (10 μl, 7.5 mg/ml in phosphate-buffered saline) was added to each well in the plate, followed by incubation at 37°C for 2 h. After incubation to dissolve the formazan crystals, 100 μl of acidified isopropanol containing 4% (vol/vol) Triton X-100 was added to each well and the optical density was measured in a kinetic microplate reader. All assays were performed in duplicate or triplicate.\n
\nTo determine the sensitivities of the primary HIV-1 isolates to drugs, phytohemagglutinin-activated peripheral blood mononuclear cells (PHA-PBMCs; 106/ml) were exposed to 50 TCID50s of each primary HIV-1 isolate and cultured in the presence or absence of various concentrations of drugs in 10-fold serial dilutions in 96-well microculture plates. To determine the drug susceptibilities of certain laboratory HIV-1 strains, MT-4 cells were used as target cells, as described previously, with minor modifications. In brief, MT-4 cells (105/ml) were exposed to 100 TCID50s of drug-resistant HIV-1 strains in the presence or absence of various concentrations of drugs and were incubated at 37°C. On day 7 of culture, the supernatant was harvested and the amount of p24 Gag protein was determined by using a fully automated chemiluminescent enzyme immunoassay system. The drug concentrations that suppressed the production of p24 Gag protein by 50% (50% inhibitory concentrations [IC50s]) were determined by comparison with the level of p24 production in drug-free control cell cultures. All assays were performed in triplicate.

Darunavir has been shown to have higher potency than saquinavir, amprenavir, nelfinavir, indinavir, lopinavir, and ritonavir in an in vitro study using MT-2 cells. The main hepatic cytochrome P450 (CYP) enzyme responsible for darunavir metabolism is CYP3A. The "boosting" dose of ritonavir increases the bioavailability of darunavir by inhibiting CYP3A.

---

Virology.Cells and Viruses. [1]
MT-4 cells are human T-lymphoblastoid cells that are highly sensitive to HIV infection, producing a rapid and strong cytopathic effect. MT4-LTR-EGFP cells are MT4 cells stably transfected with a vector containing the coding sequence for the Green Fluorescent Protein (EGFP) under control of HIV-1 Long Terminal Repeat (LTR). Upon infection of these cells with HIV, the viral transactivator protein Tat activates the LTR promoter, which in turn triggers the transcription of the EGFP coding sequence. All cells were cultured in RPMI 1640 medium supplemented with fetal calf serum and antibiotics in a humidified incubator with a 5% CO2 atmosphere at 37 °C. HIV strains used for the profiling of the compounds were wild-type HIV-1 strain IIIB and recombinant HIV strains derived from clinical isolates. Those were constructed as previously described by cotransfection of MT-4 cells with sample-derived viral protease (PR) and reverse transcriptase (RT) coding sequences and an HIV-1 HXB2-derived proviral clone deleted in the protease and RT coding region.

---

Antiviral Assays. [1]
The antiviral activity of compounds against wild-type HIV and clinical sample derived recombinant viruses was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay method as previously described. Briefly, various concentrations of the test compounds were added to wells of a flat-bottom microtiter plate. Subsequently, virus and MT-4 cells were added to a final concentration of 200−250 50% cell culture infectious doses (CCID50)/well and 30 000 cells/well, respectively. After 5 days of incubation at 37 °C with 5% CO2, the cytopathic effect (CPE) of the replicating virus was measured by the MTT method. The results of the antiviral assay were expressed as pEC50 (= −log EC50), with EC50 defined as the concentration of a compound achieving 50% CPE compared with the drug-free control. Cytotoxicity of the test compound was determined in parallel using mock-infected cell cultures containing an identical compound concentration range but no virus.

---

Generation of PI-resistant HIV-1 in vitro. [2]
MT-4 cells (105/ml) were exposed to HIV-1NL4-3 (500 TCID50s) and cultured in the presence of various PIs at an initial concentration of 0.01 to 0.03 μM. Viral replication was monitored by determination of the amount of p24 Gag produced by MT-4 cells. The culture supernatants were harvested on day 7 and were used to infect fresh MT-4 cells for the next round of culture in the presence of increasing concentrations of each drug. When the virus began to propagate in the presence of the drug, the drug concentration was generally increased two- to threefold. Proviral DNA samples obtained from the lysates of infected cells were subjected to nucleotide sequencing. This drug selection procedure was carried out until the drug concentration reached 5 μM.

---

For cytotoxicity assessment of Darunavir and PLGA-DRV, U1 macrophages were differentiated for 72 h and then treated with 6 μg/mL of DRV or PLGA-DRV for 24 h and 48 h (both with and without a BBB model). LDH activity in the cell culture was measured to evaluate cytotoxicity [3]
To determine total antioxidant capacity (TAC), differentiated U1 macrophages were treated with 6 μg/mL of DRV or PLGA-DRV for 24 h and 48 h (with or without BBB), and TAC results were normalized based on the protein level of U1 macrophages [3]
ROS activity was detected in U1 macrophages (without BBB) treated with DRV or PLGA-DRV using CM-DCFDA dye via flow cytometry (excitation/emission at 495/519 nm), and the percentage of fluorescent cells was quantified [3]
Cytokine and chemokine levels in U1 macrophages exposed to DRV, PLGA, and PLGA-DRV for 48 h (both direct treatment and with in vitro BBB) were measured using multiplex ELISA [3]
Intracellular DRV concentration was determined by treating U1 cells with 6 μg/mL of DRV or PLGA-DRV for 0.15, 0.5, 1, 4, 10, 24, 48, and 72 h, followed by LC-MS/MS analysis of intracellular DRV levels [3]
HIV-1 replication was assessed by treating U1 macrophages with 6 μg/mL of DRV or PLGA-DRV for 24 h and 48 h (with or without BBB), measuring p24 levels in the culture medium, and normalizing the data based on the protein level of U1 macrophages [3]

Animal Studies [3]
Ten twelve-week-old male and female Balb/c mice were acclimated to the animal facility for at least 7 days. Five mice per cage were housed in a sterile room with 12/12 h light–dark cycles. Temperature and humidity were maintained at a constant level in the room. There was free access to food and water. Detailed information for dosing in Balb/c mice can be found in our previous study [29]. A 2.5 mg/kg dosage of Darunavir (TMC114)/DRV or PLGA-DRV NPs was given via intranasal (IN) and intravenous (IV). For the IN group, the minimum concentration of Darunavir (TMC114)/DRV is 1.25 mg/mL to ensure that the dosing volume for each mouse is less than 2 µL per gram of mice. Given the constraints on the EE (%) of Darunavir (TMC114)/DRV in PLGA, we selected a dosage of 2.5 mg/kg, representing the highest dose achievable within the scope of this study.

---

Balb/c mice were administered Darunavir at a dose of 2.5 mg/kg either as free DRV or PLGA-DRV nanoformulation via intranasal (IN) or intravenous (IV) routes. At 1 h, 3 h, 6 h, and 12 h post-administration, brain, plasma, lung, and liver samples were collected from the mice. DRV concentrations in these tissues were quantified, and the brain-to-plasma concentration ratio was calculated (DRV concentration in brain / DRV concentration in plasma × 100%) [3]
Absorption studies of Darunavir (TMC114) were conducted in animals (specific species not specified) to evaluate its pharmacokinetic properties, with the results compared to currently approved HIV-1 protease inhibitors [1]

Absorption, Distribution and Excretion
The absolute bioavailability of a single oral dose of 600 mg darunavir is 37%, while the absolute bioavailability when combined with twice-daily 100 mg ritonavir is 82%. Studies have found that darunavir exposure is 11 times higher in patients receiving ritonavir-enhanced therapy compared to those not receiving ritonavir-enhanced therapy. The time to peak concentration (Tmax) is reached approximately 2.4 to 4 hours after oral administration. Compared to fasting, co-administration with food increases Cmax and AUC by 30% when darunavir is taken with food. A quality balance study in healthy volunteers showed that after a single dose of 400 mg 14C-darunavir (in combination with 100 mg ritonavir), approximately 79.5% and 13.9% of the radiolabeled darunavir, respectively, were excreted in feces and urine. In volunteers who did not receive ritonavir enhancement therapy, 8.0% of the dose of darunavir was excreted unchanged. In subjects who received ritonavir enhancement therapy, 48.8% of the dose of darunavir was excreted unchanged due to ritonavir's inhibition of darunavir metabolism. In volunteers who did not receive ritonavir enhancement therapy, 1.2% of the dose of darunavir was excreted unchanged in urine, compared to 7.7% in volunteers who received ritonavir enhancement therapy. In a pharmacokinetic study of darunavir in combination with ritonavir, the volume of distribution of darunavir in healthy young adult volunteers was 206.5 L (range 161.0–264.9 L). Another pharmacokinetic study showed a volume of distribution of 220 L. Darunavir has low renal clearance. Following intravenous administration, the clearance rates of darunavir alone and in combination with ritonavir (100 mg twice daily) were 32.8 L/h and 5.9 L/h, respectively. Darunavir binds to plasma proteins in approximately 95% of its components. It primarily binds to plasma α1-acid glycoprotein (AAG). After oral administration of darunavir, the time to peak absorption (Tmax) when combined with ritonavir (100 mg twice daily) is approximately 2.5–4 hours. The absolute oral bioavailability of 600 mg darunavir alone and in combination with ritonavir (100 mg twice daily) was 37% and 82%, respectively. Darunavir is distributed in rat milk; it is unknown whether the drug is excreted into human milk.
A quality balance study in healthy volunteers showed that following a single dose of 400 mg (14)C-darunavir (in combination with 100 mg ritonavir), approximately 79.5% and 13.9% of the (14)C-darunavir dose were excreted in feces and urine, respectively. Unmetabolized darunavir accounted for approximately 41.2% and 7.7% of the dose administered in feces and urine, respectively. The terminal elimination half-life of darunavir in combination with ritonavir was approximately 15 hours. Following intravenous administration, the clearance of darunavir was 32.8 L/hr when used alone and 5.9 L/hr when used in combination with 100 mg ritonavir twice daily.
For more complete data on absorption, distribution, and excretion of darunavir (of 8 items), please visit the HSDB records page.

---

Metabolism/Metabolites
Darunavir is primarily metabolized by hepatic cytochrome enzymes (mainly CYP3A). In subjects not receiving enhanced therapy, darunavir is metabolized extensively, with major pathways including carbamate hydrolysis, isobutyl aliphatic hydroxylation and aniline aromatic hydroxylation, as well as benzyl aromatic hydroxylation and glucuronidation.
In vitro human liver microsomal (HLM) experiments have shown that darunavir is primarily metabolized by oxidation. Darunavir is primarily metabolized by CYP enzymes, especially CYP3A. A mass balance study in healthy volunteers showed that after a single dose of 400 mg (14)C-darunavir (in combination with 100 mg ritonavir), most of the radioactivity in the plasma was derived from darunavir. At least three oxidative metabolites of darunavir have been identified in humans; all of these metabolites are at least 90% less active than darunavir against wild-type HIV. This study investigated the absorption, metabolism, and excretion of darunavir (a human immunodeficiency virus protease inhibitor) in eight healthy male subjects. Subjects received a single oral dose of 400 mg ((14)C) darunavir (monotherapy, no booster) or in combination with ritonavir (100 mg twice daily for 2 days before and 7 days after ritonavir administration, booster). In subjects not receiving ritonavir booster therapy, darunavir was primarily metabolized via carbamate hydrolysis, isobutyl aliphatic hydroxylation, and aniline aromatic hydroxylation, followed by benzyl aromatic hydroxylation and glucuronidation. In subjects not receiving ritonavir booster therapy, the total excretion of unchanged darunavir accounted for 8.0% of the administered dose. Ritonavir enhancement therapy significantly inhibited carbamate hydrolysis, isobutyl aliphatic hydroxylation, and aniline aromatic hydroxylation, but had no effect on benzyl aromatic hydroxylation; excretion of glucuronide metabolites was significantly increased, but still only a minor pathway. Because ritonavir inhibits darunavir metabolism, in subjects receiving ritonavir enhancement therapy, the total excretion of unchanged darunavir accounted for 48.8% of the administered dose. In subjects not receiving enhancement therapy, 1.2% of the administered dose was unmetabolized darunavir in urine, compared to 7.7% in subjects receiving enhancement therapy, indicating lower renal clearance. Darunavir is metabolized via phase I and phase II biotransformation mechanisms. Numerous metabolites were detected in vitro using animal and human hepatocytes and microsomal formulations. The metabolic pathways in rats, dogs, and humans are similar in nature. The most dominant metabolic pathway is phase I biotransformation, including carbamate hydrolysis, isobutyl aliphatic hydroxylation, and aniline aromatic hydroxylation. The metabolic pathways of dogs and humans are most similar, with both animals primarily undergoing carbamate hydrolysis. Darunavir is mainly metabolized by CYP3A. In mice and rats, darunavir treatment induces the expression of CYP3A4 in liver microsomes. Furthermore, UDP-GT activity was induced in rats. No induction was observed in dogs. Darunavir exists as a single enantiomer but does not undergo chiral conversion in vivo.

---

Biological Half-Life
When darunavir is used in combination with ritonavir, its terminal elimination half-life is approximately 15 hours.
A mass balance study in healthy volunteers showed that a single dose of 400 mg (14)C-darunavir, combined with 100 mg ritonavir… the terminal elimination half-life of darunavir combined with ritonavir was approximately 15 hours.
The pharmacokinetics of darunavir have been evaluated in vitro and in a variety of animals (mice, rats, dogs, and rabbits), which have also been used in nonclinical pharmacology and toxicology studies. The elimination half-life is rapid after oral administration, usually less than 5 hours. Animal absorption studies have shown that the pharmacokinetics of darunavir (TMC114) are comparable to those of currently approved HIV-1 protease inhibitors [1]. Darunavir (TMC114) exhibits favorable pharmacokinetic properties when used in combination with ritonavir [2]. PLGA-DRV nanoformulation improved brain delivery of darunavir in mice, with a significantly higher brain-plasma ratio compared to free DRV after intranasal administration [3].

Hepatotoxicity
A significant proportion of patients taking antiretroviral regimens containing darunavir experience some degree of elevated serum transaminases. Overall, 3% to 10% of patients experience moderate to severe elevations in serum transaminases (more than 5 times the upper limit of normal), with a higher incidence in patients co-infected with HIV-HCV. In clinical trials of darunavir, 2% to 3% of patients experienced serum ALT elevations exceeding 5 times the upper limit of normal, but no subjects developed clinically significant liver injury with jaundice. Elevated serum enzymes during treatment are usually asymptomatic and self-limiting, returning to normal with continued use. Since darunavir's approval and widespread use, there have been reports of clinically significant acute liver injury, but its clinical characteristics have not been fully described. Because most patients are taking multiple antiviral drugs concurrently, and many have chronic hepatitis B or C or non-alcoholic steatohepatitis, it is difficult to establish a causal relationship between specific anti-HIV drugs and liver injury. In reported cases, liver injury typically occurs 1 to 8 weeks after treatment, with serum enzyme elevations usually (but not always) hepatocellular. Hypersensitivity reactions (fever, rash, eosinophilia) and autoantibody formation are rare. Acute liver injury is usually self-limiting and resolves within weeks of discontinuation of darunavir. However, at least the sponsor has received reports of fatal cases and recommends monitoring liver enzymes during treatment. Finally, in patients with co-infection, initiation of highly active antiretroviral therapy with darunavir may lead to exacerbation of underlying chronic hepatitis B or C, typically occurring 2 to 12 months after treatment initiation, accompanied by hepatocellular elevations of serum enzymes and elevated serum levels of hepatitis B virus (HBV) DNA or hepatitis C virus (HCV) RNA. Darunavir treatment has not been definitively linked to lactic acidosis and acute fatty liver associated with various nucleoside analogue reverse transcriptase inhibitors. Probability score: C (Probable, rare, can lead to clinically significant liver injury).

---

Effects during pregnancy and lactation
◉ Overview of medication use during lactation
Limited information suggests that even with mothers taking up to 800 mg daily of darunavir in combination with ritonavir, drug concentrations in breast milk are extremely low or undetectable, and no adverse effects are expected on breastfed infants. Similar results are expected with darunavir in combination with cobicistat. Achieving and maintaining viral suppression through antiretroviral therapy can reduce the risk of breast milk transmission to below 1%, but not zero. For HIV-infected individuals receiving antiretroviral therapy with a persistently low viral load, breastfeeding should be supported if they choose to do so. If viral load is not suppressed, pasteurized donor breast milk or formula is recommended.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
Gynecomastia has been reported in men receiving highly effective antiretroviral therapy. Gynecomastia initially presents unilaterally, but approximately half of cases progress to bilateral gynecomastia. No changes in serum prolactin levels have been observed, and it usually resolves spontaneously within one year even with continued treatment. Some case reports and in vitro studies suggest that protease inhibitors may cause hyperprolactinemia and galactorrhea in some male patients, but this remains controversial. The implications of these findings for lactating mothers are unclear. For mothers who have established lactation, prolactin levels may not affect their ability to breastfeed.

---

Protein Binding
Darunavir binds to approximately 95% of plasma proteins. Darunavir primarily binds to plasma α1-acid glycoprotein (AAG). Darunavir (TMC114/UIC-94017) showed extremely low cytotoxicity in CD4+ MT-2 cells, with a half-maximal cytotoxic concentration (CC50) of 74 μM [2]. The PLGA-DRV nanoparticle formulation did not induce cytotoxicity in U1 macrophages (LDH activity remained unchanged after treatment) nor did it elicit an inflammatory response in HIV-infected U1 macrophages [3].

[1]. Discovery and selection of TMC114, a next generation HIV-1 protease inhibitor. J Med Chem. 2005 Mar 24;48(6):1813-22.

[2]. Novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI) UIC-94017 (TMC114) with potent activity against multi-PI-resistant human immunodeficiency virus in vitro. Antimicrob Agents Chemother. 2003 Oct;47(10):

[3]. Darunavir Nanoformulation Suppresses HIV Pathogenesis in Macrophages and Improves Drug Delivery to the Brain in Mice. Pharmaceutics . 2024 Apr 19;16(4):555.

Darunavir is an N,N-disubstituted benzenesulfonamide compound with an unsubstituted amino group at the 4-position, used to treat HIV infection. As a second-generation HIV protease inhibitor, darunavir is designed to form a potent interaction with the proteases of various HIV strains, including strains from previously treated patients with multiple resistance mutations to other protease inhibitors. It is both an HIV protease inhibitor and an antiviral drug. It is a furan, carbamate, and sulfonamide compound. Darunavir (brand name: pregabalin) is a prescription drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV infection in adults and children. Darunavir must be used in combination with pharmacokinetic enhancers (ritonavir (brand name: Noval) or cobicistat (brand name: Tebostat)) and other anti-HIV drugs. When darunavir is used in combination with ritonavir, it can be used in adults weighing at least 10 kg (22 lbs) and children aged 3 years and older. When used in combination with cobicistat, darunavir can be used in adults and children weighing at least 40 kg (88 lbs) and meeting specific criteria, as determined by a healthcare provider. (A fixed-dose combination tablet containing darunavir and cobicistat is also available [brand name: Prescobis].) Darunavir is a protease inhibitor used in combination with other anti-HIV protease inhibitors and ritonavir to effectively treat HIV-1 infection. As a second-generation protease inhibitor, darunavir is designed to combat resistance to standard HIV therapies. Darunavir was initially approved by the U.S. Food and Drug Administration (FDA) in 2006. Its potential as a treatment for SARS-CoV-2 (the coronavirus that causes COVID-19) is currently being investigated due to in vitro experimental evidence demonstrating its effectiveness against SARS-CoV-2 infection. Clinical trials are ongoing and are expected to conclude in August 2020. Darunavir is a protease inhibitor. Its mechanism of action is as an HIV protease inhibitor, a cytochrome P450 3A inhibitor, and a cytochrome P450 2D6 inhibitor. Darunavir is an antiretroviral protease inhibitor used to treat and prevent human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). Darunavir can cause a transient increase in serum transaminase levels, usually asymptomatic, and is associated with rare cases of clinically significant acute liver injury. In patients with co-infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), highly active antiretroviral therapy with darunavir may exacerbate pre-existing chronic hepatitis B or C. Darunavir is a non-peptide inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease with anti-HIV activity. After oral administration, darunavir selectively targets and binds to the active site of the HIV-1 protease, inhibiting the dimerization and catalytic activity of the HIV-1 protease. This inhibits the proteolytic cleavage of viral Gag and Gag-Pol polymers in HIV-infected cells. This inhibition results in the production of immature, non-infectious viral proteins that cannot form mature viral particles, thus preventing HIV replication. Darunavir is an HIV protease inhibitor used to treat AIDS and HIV infection. Because antiretroviral resistance can develop when used alone, it needs to be used in combination with other anti-HIV drugs.

---

Drug Indications
Darunavir, in combination with ritonavir and other antiretroviral drugs, is indicated for the treatment of HIV-1 infection in children aged 3 years and older and adults.
FDA Label
Darunavir, in combination with low-dose ritonavir, is indicated for the treatment of patients with HIV-1 infection in combination with other antiretroviral drugs (see Section 4.2). Darunavir (Mylan) tablets in 75 mg, 150 mg, 300 mg, and 600 mg doses are available to provide appropriate dosing regimens (see Section 4.2): for the treatment of adult HIV-1 infected patients who have previously received antiretroviral therapy (ART), including those who have received extensive prior treatment; and for the treatment of children aged 3 years and older with HIV-1 infection who weigh at least 15 kg. When deciding to initiate treatment with darunavir in combination with low-dose ritonavir, the patient's treatment history and drug-related mutation patterns should be carefully considered. Genotypic or phenotypic testing (if available) and treatment history should guide the use of darunavir (see Sections 4.2, 4.4, and 5.1). Darunavir in combination with low-dose ritonavir is indicated for the treatment of human immunodeficiency virus (HIV-1) infected patients in combination with other antiretroviral agents. Darunavir, in combination with cobicistat, is indicated for the treatment of human immunodeficiency virus (HIV-1) infection in adults and adolescents (12 years and older, weighing at least 40 kg) in combination with other antiretroviral agents (see Section 4.2). Darunavir Mylan 400 mg and 800 mg tablets are available to provide an appropriate dosing regimen for adults and children aged 3 years and older, weighing at least 40 kg, to treat HIV-1 infection in patients who have not received prior antiretroviral therapy (ART) (see Section 4.2). Darunavir should be considered in patients who have previously received antiretroviral therapy (ART) and do not have darunavir resistance-associated mutations (DRV-RAMs) if their plasma HIV-1 RNA is < 100,000 copies/mL and their CD4+ cell count is ≥ 100 × 10⁶ cells/L. When deciding to initiate darunavir treatment in patients who have previously received ART, genotyping results should be used as a guide (see Sections 4.2, 4.3, 4.4, and 5.1). Darunavir Krka (400 mg and 800 mg) in combination with low-dose ritonavir is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents. Darunavir Krka 400 mg and 800 mg tablets may be used to provide an appropriate dosing regimen for HIV-1-infected adults and children aged 3 years and older weighing at least 40 kg who meet the following criteria: have not received antiretroviral therapy (ART) (see Section 4.2); or have received ART but have no darunavir resistance-associated mutations (DRV-RAMs), and have plasma HIV-1 RNA <100,000 copies/mL and a CD4+ cell count ≥100 × 10⁶ cells/L. For patients who have previously received ART, genotyping results should guide the decision to initiate darunavir treatment (see Sections 4.2, 4.3, 4.4, and 5.1). Darunavir Keka 600 mg tablets, in combination with low-dose ritonavir, are indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents. Darunavir Keka 600 mg tablets can be used to provide an appropriate dosing regimen (see Section 4.2): for the treatment of adult HIV-1 infected patients who have previously received antiretroviral therapy (ART), including those who have received extensive prior treatment; and for the treatment of children aged 3 years and older with HIV-1 infection weighing at least 15 kg. When deciding to initiate darunavir in combination with low-dose ritonavir, the patient's previous treatment history and drug-related mutation patterns should be carefully considered. Genotyping or phenotyping (if available) and treatment history should guide the use of darunavir. Pregabalin (PREZISTA) in combination with low-dose ritonavir is indicated for the treatment of HIV-1 infection in adults and children aged 3 years and older, weighing at least 15 kg, in combination with other antiretroviral agents. Pregabalin (PREZISTA) in combination with cobicistat is indicated for the treatment of HIV-1 infection in adults and adolescents (12 years and older, weighing at least 40 kg), in combination with other antiretroviral agents. When deciding to initiate treatment with pregabalin (PREZISTA) in combination with cobicistat or low-dose ritonavir, the patient's treatment history and drug-related mutation patterns should be carefully considered. Genotypic or phenotypic testing (if available) and treatment history should guide the use of pregabalin (PREZISTA). Pregabalin in combination with low-dose ritonavir is indicated for the treatment of HIV-1 infection in patients, in combination with other antiretroviral agents. Pregabalin tablets in 75 mg, 150 mg, and 600 mg formulations are available to provide appropriate dosing regimens for the treatment of HIV-1 infection in adults who have received prior antiretroviral therapy (ART), including those who have received extensive prior treatment; and for the treatment of HIV-1 infection in children aged 3 years and older and weighing at least 15 kg. When deciding to initiate treatment with pregabalin in combination with low-dose ritonavir, the patient's treatment history and drug-related mutation patterns should be carefully considered. Genotypic or phenotypic testing (if available) and treatment history should guide the use of pregabalin (PREZISTA). PREZISTA in combination with low-dose ritonavir is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents. PREZISTA in combination with cobicistat is indicated for the treatment of HIV-1 infection in adults and adolescents (12 years and older and weighing at least 40 kg) in combination with other antiretroviral agents. PREZISTA 400 mg and 800 mg tablets are indicated for use in adults and children aged 3 years and older with HIV-1 infection, weighing at least 40 kg, who meet the following criteria: no prior antiretroviral therapy (ART); prior ART without darunavir resistance-associated mutations (DRV RAM); and plasma HIV-1 RNA < 100 mmol/L. Viral load is 100,000 copies/mL, and CD4+ cell count is ≥ 100 × 10⁶ cells/L. For these prior ART patients, genotyping results should guide the decision to initiate PREZISTA treatment. 400 mg and 800 mg film-coated tablets are also available. Darunavir (Krka dd) in combination with low-dose ritonavir is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents. Darunavir (Krka dd) in combination with cobicistat is indicated for the treatment of HIV-1 infection in adults in combination with other antiretroviral agents (see Section 4.2). Darunavir Krka double-dose tablets (400 mg and 800 mg) can be used to provide an appropriate dosing regimen for HIV-1 infection in adults and children aged 3 years and older and weighing at least 40 kg, provided that these patients meet the following criteria: have not received antiretroviral therapy (ART) (see Section 4.2); or have received ART but have no darunavir resistance-associated mutations (DRV-RAMs), and have plasma HIV-1 RNA <100,000 copies/mL and CD4+ cell count ≥100 × 10⁶ cells/L. For patients who have previously received ART, genotyping results should guide the decision to initiate darunavir treatment (see Sections 4.2, 4.3, 4.4, and 5.1). Darunavir (Krka dd) 600 mg film-coated tablets, used in combination with low-dose ritonavir, are indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents. Darunavir (Krka dd) 600 mg tablets can be used to provide an appropriate dosing regimen (see Section 4.2): for the treatment of adults with HIV-1 infection who have previously received antiretroviral therapy (ART), including those who have received extensive prior treatment; and for the treatment of children aged 3 years and older with HIV-1 infection who weigh at least 15 kg. When deciding to initiate darunavir in combination with low-dose ritonavir, the patient's previous treatment history and drug-related mutation patterns should be carefully considered. Genotyping or phenotyping (if available) and treatment history should guide the use of darunavir.
Treatment of Human Immunodeficiency Virus (HIV-1) Infection

---

Mechanism of Action
HIV-1 protease is essential for the processing of viral precursor proteins and viral maturation, preparing for infection, and is therefore a target of HIV antiretroviral therapy. Protease inhibitors are part of highly active antiretroviral therapy (HAART) for HIV-infected patients. Studies have shown that HAART can effectively suppress the virus and significantly reduce morbidity and mortality. Darunavir is an HIV protease inhibitor that blocks HIV replication by binding to the protease, thereby inhibiting the dimerization and catalytic activity of the HIV-1 protease. Specifically, it inhibits the cleavage of the HIV-encoded Gag-Pol protein in virus-infected cells, preventing the formation of mature viral particles and thus preventing the spread of infection. Darunavir's tight contact with the primary chain of amino acids (Asp-29 and Asp-30) at the protease's active site may be one of the reasons for its potency and efficacy against drug-resistant HIV-1 variants. Darunavir is known to bind to different sites on enzymes: the active site cavity and the surface of a flexible valve in the protease dimer. Due to its molecular flexibility, darunavir can adapt to changes in protease shape. Darunavir, as a protease inhibitor, inhibits the cleavage of the HIV-encoded gag-pol polyprotein in virus-infected cells, thereby preventing the formation of mature and infectious new viral particles. It was selected for its efficacy against wild-type HIV-1 and HIV strains resistant to currently approved protease inhibitors. Darunavir is an inhibitor of the HIV-1 protease. It selectively inhibits the cleavage of the HIV-encoded Gag-Pol polyprotein in infected cells, thereby preventing the formation of mature viral particles.

---

Compared to ampravir, TMC126 (an analog of darunavir) has increased affinity for HIV-1 protease, mainly due to the formation of additional hydrogen bonds with the protein backbone in the P2 pocket; the modification of the benzenesulfonamide P2' substituent of TMC126 establishes a structure-activity relationship (SAR), and darunavir (TMC114) shows similar antiviral activity against both wild-type and mutant HIV-1. The additional hydrogen bonds in the P2 pocket are not the only reason for the good antiviral properties of darunavir [1]
Darunavir (TMC114/UIC-94017) contains a 3(R),3a(S),6a(R)-bis(tetrahydrofuran carbamate) (bis-THF) and a sulfonamide isostere. Due to its potent activity and good pharmacokinetic properties when used in combination with ritonavir, it is a potential therapeutic drug for primary and multiprotease inhibitor-resistant HIV-1 infection [2]. Antiretroviral therapy (ART) can inhibit peripheral HIV, but due to insufficient concentration of ART drugs in the brain, it cannot adequately treat neurological HIV. PLGA-DRV nanoparticles, administered intranasally, overcome the limitations of drug metabolic stability and blood-brain barrier permeability, providing a promising method for treating HIV-related neurological diseases [3].

C27H37N3O7S

547.660

547.235

C, 59.21; H, 6.81; N, 7.67; O, 20.45; S, 5.85

206361-99-1

Darunavir Ethanolate;635728-49-3;Darunavir-d9;1133378-37-6; 2281870-65-1 (dihydrate); 635728-49-3 (ethanolate); 206361-99-1 (free)

213039

White to off-white solid powder

1.3±0.1 g/cm3

74-76ºC

1.620

3.94

3

9

12

38

853

5

O=C(O[C@@H]1[C@@]2([H])[C@@](OCC2)([H])OC1)N[C@@H](CC3=CC=CC=C3)[C@H](O)CN(S(=O)(C4=CC=C(N)C=C4)=O)CC(C)C

CJBJHOAVZSMMDJ-HEXNFIEUSA-N

InChI=1S/C27H37N3O7S/c1-18(2)15-30(38(33,34)21-10-8-20(28)9-11-21)16-24(31)23(14-19-6-4-3-5-7-19)29-27(32)37-25-17-36-26-22(25)12-13-35-26/h3-11,18,22-26,31H,12-17,28H2,1-2H3,(H,29,32)/t22-,23-,24+,25-,26+/m0/s1

[(3aS,4R,6aR)-2,3,3a,4,5,6a-hexahydrofuro[2,3-b]furan-4-yl] N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenylbutan-2-yl]carbamate

Darunavir; TMC-114; TMC114; TMC 114; UIC-94017; Darunavir; 206361-99-1; Darunavirum; UIC 94017; UIC94017; Trade name: Prezista

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)