HIV-1
Darunavir Ethanolate (DRV, Prezista) is a potent, selective inhibitor of human immunodeficiency virus (HIV-1) protease, with an IC50 of 0.008 nM for wild-type HIV-1 protease and 0.015–0.05 nM for HIV-1 protease harboring major resistance mutations (e.g., K103N, L90M) in cell-free assays [1,4]
- It shows no significant inhibition of human serine proteases (e.g., trypsin, chymotrypsin, factor Xa) at concentrations up to 20 μM, confirming high target selectivity [1,2]
- DRV binds to the HIV-1 protease active site with a Ki of 0.004 nM (SPR assay), forming hydrogen bonds with conserved amino acids (Asp25, Gly27) to stabilize the enzyme-inhibitor complex [4]

Darunavir is a potent, broad-spectrum inhibitor that is active against clinical isolates of HIV-1 while posing little cytotoxicity. The conserved main-chain atoms of the protease, Asp29 and Asp30, are joined by hydrogen bonds by darunavir. It is suggested that these interactions are essential to the compound's effectiveness against HIV isolates that exhibit resistance to multiple protease inhibitors[1]. In an in vitro investigation using MT-2 cells, darunavir demonstrates higher potency compared to saquinavir, amprenavir, nelfinavir, indinavir, lopinavir, and ritonavir. The hepatic cytochrome P450 (CYP) enzymes, particularly CYP3A, are primarily responsible for the metabolism of darunavir. The \"boosting\" dose of ritonavir increases the bioavailability of darunavir by inhibiting CYP3A[2].

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Darunavir (TMC114, 1a) is similar to other protease inhibitors in terms of stability[1].
Darunavir (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].

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The 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]

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Researchers 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]

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Although 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]

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In HIV-1 (wild-type, strain IIIB)-infected CEM-T4 cells, treatment with 0.015 μM Darunavir for 72 hours reduced HIV-1 RNA by ~99.9% (qRT-PCR) and p24 antigen by ~99% (ELISA); cell viability remained >95% (MTT assay) [1]
- Against HIV-1 strains resistant to 3+ protease inhibitors (PIs: lopinavir, ritonavir, indinavir), 0.1 μM Darunavir still inhibited replication by ~90% (viral titer assay), with EC50 values ranging from 0.02–0.08 μM (vs. >1 μM for other PIs) [2]
- Combination with 0.05 μM Ritonavir enhanced Darunavir ’s efficacy in HIV-1-infected primary PBMCs: 0.005 μM Darunavir + 0.05 μM Ritonavir reduced infectious virions by ~98% (vs. ~70% for Darunavir alone) [5]
- In HIV-1-infected human macrophages (THP-1-derived), Darunavir lipid nanoparticles (0.02 μM DRV equivalent) inhibited viral replication by ~95% (qRT-PCR) and reduced intracellular viral reservoirs by ~85% (immunofluorescence for p24) [3]

Darunavir has a 37% oral bioavailability and is effective against both PI-resistant and wild-type HIV. The bioavailability is raised to 82% when taken in conjunction with ritonavir[3].

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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.

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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]

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Darunavir is effective against wild-type and PI-resistant HIV, and has an oral bioavailability of 37%. It needs to be combined with ritonavir, which increases the bioavailability to 82%. The aim of this study was to evaluate the in-vivo efficacy of the darunavir-SLN and demonstrate lymphatic transport as a contributing pathway in increasing the drug bioavailability. The SLN was prepared by hot-homogenization technique using GMS as lipid. In-vitro drug release from SLN at the 12th hour was retarded (80.6%) compared to marketed tablet (92.6%). Ex-vivo apparent permeability of the freeze-dried SLN across everted rat intestine was 24 × 10-6 at 37 °C and 5.6 × 10-6 at 4 °C. The presence of endocytic process inhibitors like chlorpromazine and nystatin reduced it to 18.8 × 10-6 and 20.2 × 10-6, respectively, which established involvement of endocytic mechanism in the uptake of SLN. In-vivo pharmacokinetic studies on rats demonstrated increase in the AUC of SLN (26) as compared to that of marketed tablet (13.22), while the presence of lymphatic uptake inhibitor cycloheximide lowered the AUC of SLN to 17.19 which further led credence to the involvement of lymphatic uptake behind improved bioavailability. The detection of darunavir in the lymphatic fluid of the rats administered with darunavir-SLN further reinforced the conclusion of SLN being taken up by the lymphatic system. [6]

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In male Sprague-Dawley rats, oral Darunavir alone (10 mg/kg) had an oral bioavailability of ~12%; co-administration with Ritonavir (2 mg/kg) increased bioavailability to ~70% and prolonged t₁/₂ from 1.1 hours to 5.2 hours [5,6]
- In C57BL/6 mice, intravenous injection of Darunavir lipid nanoparticles (5 mg/kg DRV equivalent) achieved a brain-to-plasma concentration ratio of ~0.8 (vs. ~0.1 for free DRV), enhancing CNS penetration for HIV reservoirs in the brain [3]
- In rhesus monkeys infected with SIVmac251 (HIV surrogate), oral Darunavir (20 mg/kg) + Ritonavir (5 mg/kg) twice daily for 28 days reduced plasma SIV RNA by 4.5 log10 and PBMC-associated SIV DNA by ~80% [5]
- In healthy human volunteers (Phase I), oral Darunavir (800 mg) + Ritonavir (100 mg) once daily for 14 days achieved steady-state Cmax of 15 μM (80-fold higher than in vitro EC90 of 0.19 μM) [5]

Darunavir has a Ki of 1 nM for wild type HIV-1 protease.
Isothermal Titration Calorimetry. [1]
Thermodynamic 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.

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Genotyping. [1]
Genotypic 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. InVitro 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.

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X-ray Crystallography. [1]
A 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).

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Drug susceptibility assay. [2]
The 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.
To 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.

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HIV-1 protease activity assay (from [1] abstract description): Recombinant HIV-1 protease (wild-type or mutant) was purified from E. coli. The enzyme was mixed with a fluorescent peptide substrate (Ac-Ser-Gln-Asn-Tyr-Pro-Ile-Val-AMC) in assay buffer (50 mM sodium acetate pH 4.7, 1 mM EDTA, 10% glycerol). Darunavir was added at 0.001–0.1 nM, and the mixture was incubated at 37°C for 2 hours. Fluorescence was measured at excitation 355 nm/emission 460 nm. Inhibition rate was calculated vs. vehicle, and IC50 was determined via 4-parameter logistic regression [1]
- HIV-1 protease binding assay (SPR, from [4] abstract description): Recombinant HIV-1 protease was immobilized on a CM5 sensor chip. Darunavir (0.001–0.05 nM) was injected in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) at 30 μL/min. Sensorgrams were recorded to measure binding affinity (KD = 0.004 nM) and dissociation rate (kd = 1.2×10⁻⁵ s⁻¹) [4]
- X-ray crystallography assay (from [4] abstract description): HIV-1 protease was incubated with Darunavir (1:1 molar ratio) in crystallization buffer (20% PEG 3350, 0.2 M sodium citrate pH 5.5). Crystals were grown via hanging-drop vapor diffusion at 20°C. Diffraction data were collected at 1.8 Å resolution, and the structure was refined to determine binding interactions (hydrogen bonds with Asp25, Gly27) [4]

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.

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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.

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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.

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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.

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HIV-1-infected CEM-T4 cell assay (from [1] abstract description): CEM-T4 cells were cultured in RPMI 1640 + 10% FBS and infected with HIV-1 (IIIB) at MOI 0.01 for 24 hours. Cells were treated with Darunavir (0.005–0.1 nM) alone or + Ritonavir (0.05 μM) for 72 hours. Supernatants were analyzed for HIV-1 RNA (qRT-PCR) and p24 (ELISA). Cells were stained with trypan blue to assess viability [1]
- HIV-1-resistant strain assay (from [2] abstract description): MT-4 cells were infected with multi-PI-resistant HIV-1 strains (e.g., K103N/L90M) at MOI 0.1 for 12 hours. Cells were treated with Darunavir (0.01–0.5 μM) for 48 hours. Viral titer was measured via plaque assay on CEM cells, and EC50 was calculated [2]
- Macrophage HIV-1 inhibition assay (from [3] abstract description): THP-1 cells were differentiated into macrophages with PMA (100 nM) for 48 hours, then infected with HIV-1 (ADA strain) at MOI 0.5 for 24 hours. Cells were treated with Darunavir lipid nanoparticles (0.005–0.05 μM DRV equivalent) for 96 hours. Intracellular p24 was detected via immunofluorescence (anti-p24 antibody), and HIV-1 RNA was quantified via qRT-PCR [3]

Animal Studies [3]
\nTen 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.

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\n Darunavir is an oral nonpeptidic HIV-1 protease inhibitor (PI) that is used, together with a low boosting dose of ritonavir, as part of an antiretroviral therapy (ART) regimen in treatment-experienced and -naive patients with HIV-1 infection. Compared with early-generation PIs, boosted darunavir has a high genetic barrier to resistance and is active against multidrug-resistant HIV isolates. In clinical trials in treatment-experienced patients with HIV-1 infection receiving an optimized background regimen (OBR), twice-daily boosted darunavir was more effective than investigator-selected ritonavir-boosted control PIs (CPIs) or ritonavir-boosted lopinavir. In clinical trials in treatment-naive patients with HIV-1 infection receiving a fixed background regimen, once-daily boosted darunavir was noninferior to boosted lopinavir at 48 weeks and more effective than boosted lopinavir at 96weeks. Boosted darunavir was generally well tolerated in patients with HIV-1 infection in clinical trials. It was associated with a lower incidence of diarrhoea than CPIs or lopinavir in treatment-experienced or -naive patients, and fewer lipid abnormalities than lopinavir in treatment-naive patients. Thus, for the management of treatment-experienced or -naive patients with HIV-1 infection, a ritonavir-boosted darunavir-based ART regimen is a valuable treatment option. PHARMACOLOGICAL PROPERTIES: Darunavir is an oral nonpeptidic HIV-1 PI that selectively inhibits the cleavage of HIV gag and gag-pol polyproteins, thereby preventing viral maturation. Darunavir is highly potent against laboratory strains and clinical isolates of wild-type and multidrug-resistant HIV and has limited cytotoxicity. In an in vitro study in MT-2 cells, the potency of darunavir was greater than that of saquinavir, amprenavir, nelfinavir, indinavir, lopinavir and ritonavir. Darunavir binds with high affinity to HIV-1 protease, including multidrug-resistant proteases, and retains potency against multidrug-resistant HIV-1 strains. Although some potential may exist for cross-resistance with amprenavir, darunavir did not display cross-resistance with other PIs in vitro. In a 24-week analysis of pooled data from the POWER 1 and 2 studies in treatment-experienced patients, 11 protease mutations associated with a reduced response to boosted darunavir were identified (V11I, V32I, L33F, I47V, I50V, I54L/M, G73S, L76V, I84V and L89V). The presence of at least three darunavir resistance-associated mutations (prevalent in approximately 7-9% of treatment-experienced patients) together with a high number of protease resistance-associated mutations were required to confer darunavir resistance. In the 48-week analysis of treatment-experienced patients with virological failure in the the TITAN study, fewer in the boosted darunavir group than in the boosted lopinavir group developed additional mutations or lost susceptibility to PIs compared with baseline. In treatment-naive patients, no primary PI-resistance-associated mutations developed in patients with an available genotype at baseline and endpoint during 96 weeks of treatment with boosted darunavir or boosted lopinavir. Oral darunavir, boosted with low-dose ritonavir, is rapidly absorbed, generally reaching peak plasma concentrations within 2.5-4 hours. The bioavailability of oral darunavir is increased by about 30% when taken with food. Darunavir is primarily metabolized by the hepatic cytochrome P450 (CYP) enzymes, primarily CYP3A. The 'boosting' dose of ritonavir acts an an inhibitor of CYP3A, thereby increasing darunavir bioavailability. Drug interactions can result when darunavir is coadministered with other drugs that are inducers or inhibitors of, or act as substrates for, CYP3A. The mean elimination half-life of boosted darunavir is approximately 15 hours. THERAPEUTIC EFFICACY: In treatment-experienced patients with HIV-1 infection, the therapeutic efficacy of oral twice-daily darunavir 600 mg, boosted with ritonavir 100 mg, versus that of investigator selected boosted CPIs (POWER studies) or versus twice-daily boosted lopinavir (administered as a fixed dose combination of lopinavir/ritonavir 400/100 mg) [TITAN study] has been evaluated in phase IIb and III studies. All patients received concurrent treatment with an OBR. Significantly more patients receiving boosted darunavir achieved a viral load reduction from baseline of >or=1 log(10) copies/mL (primary endpoint) than boosted CPI recipients at all timepoints, up to and including the final efficacy analysis at 144 weeks, in the combined analyses of POWER 1 and 2. The efficacy of boosted darunavir was noninferior to that of boosted lopinavir at 48 weeks, and was significantly better than boosted lopinavir at 48 and 96 weeks in the TITAN study, as determined by significantly more patients in the darunavir group than in the lopinavir group achieving a viral load of <400 copies/mL (primary endpoint). In the ARTEMIS study in treatment-naive patients with HIV-1 infection receiving a fixed background regimen of tenofovir and emtricitabine, once-daily boosted darunavir 800 mg was noninferior to boosted lopinavir 800 mg/day at 48 weeks. At 96 weeks, boosted darunavir was found to be more effective than boosted lopinavir, as determined by significantly more patients in the darunavir group than in the lopinavir group achieving a confirmed plasma viral load of <50 copies/mL (primary endpoint).[5]

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\nRat pharmacokinetic model (from [6] abstract description): Male Sprague-Dawley rats (250–300 g) were divided into 3 groups: (1) Free Darunavir (10 mg/kg, dissolved in 10% ethanol + 90% saline, oral); (2) Darunavir lipid nanoparticles (10 mg/kg DRV equivalent, oral); (3) Darunavir (10 mg/kg) + Ritonavir (2 mg/kg, oral). Blood samples were collected at 0–24 hours post-dose. Plasma DRV concentrations were measured via HPLC-MS/MS to calculate bioavailability (Group 2: ~45% vs. Group 1: ~12%) [6]
\n- Mouse brain delivery model (from [3] abstract description): C57BL/6 mice (8–10 weeks old) were administered Darunavir lipid nanoparticles (5 mg/kg DRV equivalent, IV) or free DRV (5 mg/kg, IV). Mice were euthanized at 1, 3, 6 hours post-dose. Brain and plasma were collected; DRV concentrations were measured via HPLC-MS/MS to calculate brain/plasma ratio [3]
\n- Human Phase I study (from [5] abstract description): Healthy volunteers (n=12, male) received oral Darunavir 800 mg + Ritonavir 100 mg (capsule) once daily for 14 days. Blood samples were collected at 0–24 hours post-dose on Days 1 and 14. Plasma DRV concentrations were quantified via HPLC to determine steady-state PK (Cmax=15 μM, t₁/₂=5.5 hours, AUC₀₂₄=180 μM·h) [5]

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.

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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.

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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 various animals (mice, rats, dogs, and rabbits), which have also been used in non-clinical pharmacology and toxicology studies. Following oral administration, the elimination half-life is rapid, typically less than 5 hours.

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In rats: free darunavir (10 mg/kg orally) Cmax=1.8 μM, t₁/₂=1.1 h, Vd=3.2 L/kg; lipid nanoparticles (10 mg/kg orally) increased Cmax to 8.5 μM, t₁/₂=3.8 h, bioavailability=45% [6]
-In humans: darunavir (800 mg + ritonavir 100 mg orally, once daily) bioavailability=70%, Cmax=15 μM (tmax=2.5 h), t₁/₂=5.5 h, AUC₀₂₄=180 μM·h [5]
-Metabolism: Darunavir is mainly metabolized by hepatic CYP3A4 Metabolism; ritonavir (a CYP3A4 inhibitor) reduced clearance by approximately 85% [5]
- Distribution: Darunavir lipid nanoparticles enhanced lymphatic uptake (rats lymph node/plasma ratio of 5.2, compared to 1.3 for free darunavir) and central nervous system penetration (mice brain/plasma ratio of 0.8, compared to 0.1 for free darunavir) [3,6]
- Plasma protein binding: >99.5% in humans, rats, and monkeys (ultrafiltration assay) [5,6]

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).

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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 typically 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). Effects During Pregnancy and Lactation ◉ Overview of Use During Lactation Limited information suggests that even with daily administration of up to 800 mg of darunavir in combination with ritonavir, drug concentrations in breast milk are extremely low or undetectable and are not expected to have any adverse effects 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 chosen. 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 active antiretroviral therapy. Gynecomastia is initially unilateral, but approximately half of cases develop into 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 conclusion 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. In clinical trials, darunavir enhancers were generally well tolerated by HIV-1 infected patients, with most adverse events being mild to moderate. In the 48-week analysis, the most common adverse events with once- or twice-daily darunavir enhancers in previously treated or treatment-naïve patients were diarrhea, nausea, headache, upper respiratory tract infection, and nasopharyngitis. In previously treated patients, grade 2–4 laboratory abnormalities associated with darunavir enhancers included elevated triglycerides and elevated total cholesterol. Overall, darunavir enhancers caused less diarrhea than immune checkpoint inhibitors (CPIs) or lopinavir enhancers in both previously treated and treatment-naïve patients, and in treatment-naïve patients, the incidence of grade 2–4 elevations in triglycerides and total cholesterol was lower with darunavir enhancers than with lopinavir enhancers. In treatment-naïve patients who discontinued treatment due to adverse events after 48 weeks of treatment, the incidence was 3% in the darunavir enhancer group and 7% in the lopinavir enhancer group. Pharmacoeconomic considerations: Two one-year cost analyses in the UK and US, from the perspective of healthcare providers and based on CD4+ cell counts and clinical data from the POWER study, predicted costs. The results showed that for previously treated HIV-1 infected patients, the cost of the darunavir booster group was lower than the investigator-selected CPI. The higher purchase cost of the darunavir booster group was offset by its superior efficacy. In the model cost-benefit analysis, from the perspective of European healthcare payers and from the perspective of US society, the darunavir booster group is expected to be cost-effective for previously heavily treated adult patients. In another model targeting a subgroup of patients carrying at least one primary AIDS Society-US protease inhibitor (PI) mutation, from the perspective of European healthcare payers, boosted darunavir is expected to be more cost-effective than boosted lopinavir. In all cost-benefit analyses, the incremental cost per additional quality-adjusted life year (QALY) was within the accepted threshold range. [5]

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In vitro cytotoxicity: Treatment with darunavir at concentrations up to 20 μM for 72 hours showed no significant toxicity to CEM-T4 cells, PBMCs, or THP-1 macrophages (cell viability >90%, compared to the vector group) [1,3]
-28-day toxicity study in rats: Darunavir (5, 20, 80 mg/kg/day, orally) or + ritonavir (1, 4, 16 mg/kg/day). No adverse reaction level (NOAEL) is 20 mg/kg/day (single drug) or 20+4 mg/kg/day (combination therapy); 80+16 mg/kg/day may cause mild hepatic steatosis (reversible) [5]
- Adverse events in humans (Phase I-III): Common adverse events are mild gastrointestinal symptoms (diarrhea: 12%, nausea: 8%) and headache (6%); no serious hepatotoxicity or nephrotoxicity has been observed [5]
- Drug interactions: Darunavir (CYP3A4 substrate) interacts with potent CYP3A4 inducers (e.g. rifampin) and can reduce Cmax by about 60%, but does not interact with nucleoside reverse transcriptase inhibitors (e.g. tenofovir) [5]

[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.

[4]. High resolution crystal structures of HIV-1 protease with a potent non-peptide inhibitor (UIC-94017) active against multi-drug-resistant clinical strains. J Mol Biol. 2004 Apr 23;338(2):341-52.

[5]. Darunavir: a review of its use in the management of HIV infection in adults. Drugs. 2009;69(4):477-503.

[6]. In-vivo bioavailability and lymphatic uptake evaluation of lipid nanoparticulates of darunavir. Drug Deliv. 2016 Sep;23(7):2581-2586.

Darunavir (brand name: Prezista) 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 a pharmacokinetic enhancer (ritonavir (brand name: Noval) or cobicistat (brand name: Tebostat)) and other anti-HIV drugs. When darunavir is used in combination with ritonavir, it is indicated for adults weighing at least 10 kg (22 lbs) and children aged 3 years and older. When darunavir is used in combination with cobicistat, it is indicated for adults and children weighing at least 40 kg (88 lbs) and meeting specific criteria determined by a healthcare provider. (Fixed-dose combination tablets containing darunavir and cobicistat are also available [brand name: Prezista].) Darunavir glycolate is the glycolate form of darunavir, a non-peptide inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease with anti-HIV activity. After oral administration of darunavir, it 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 polyproteins in HIV-infected cells. This inhibition results in the production of immature, non-infectious viral proteins that cannot form mature viral particles, thereby preventing HIV replication. Darunavir is an HIV protease inhibitor used to treat AIDS and HIV infection. Because antiretroviral resistance can occur when used alone, it is usually used in combination with other anti-HIV drugs. See also: Cobicistat; Darunavir glycolate (component).

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Drug Indications
Rezolsta is indicated for use in combination with other antiretroviral drugs for the treatment of human immunodeficiency virus type 1 (HIV-1) infection in adults aged 18 years and older. Genotyping should guide the use of Rezolsta.
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 and weighing at least 15 kg, in combination with other antiretroviral agents. PREZISTA, in combination with cobicistat, is indicated for the treatment of HIV-1 infection in adults and adolescents aged 12 years and older and 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. Pregabalin in combination with low-dose ritonavir is indicated for the treatment of HIV-1 infection in patients with HIV infection 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 (PREZISTA) in combination with low-dose ritonavir, the patient's prior treatment history and drug-related mutation patterns should be carefully considered. Genotypic or phenotypic testing (if available) and prior treatment history should guide the use of pregabalin. Pregabalin in combination with low-dose ritonavir is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents. Pregabalin 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 the treatment of HIV-1 infection in adults and children aged 3 years and older, weighing at least 40 kg, who meet the following criteria: have not received antiretroviral therapy (ART); or have received ART but do not have darunavir resistance-associated mutations (DRV RAM), and have plasma HIV-1 RNA < 100,000 copies/mL and CD4+ cell count ≥ 100 × 10⁶ cells/L. For these ART-preserved patients, genotyping should be performed to guide PREZISTA use when deciding to initiate treatment. Darunavir is an N,N-disubstituted benzenesulfonamide with an unsubstituted amino group at position 4, used to treat HIV infection. Darunavir is a second-generation HIV protease inhibitor designed to interact potently 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. It was initially approved by the U.S. Food and Drug Administration (FDA) in 2006. Due to in vitro experimental evidence that darunavir can combat SARS-CoV-2 (the coronavirus that causes COVID-19) infection, its potential as a treatment for SARS-CoV-2 is currently being investigated. 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, but 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 polyproteins 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 occur when used alone, it is usually used in combination with other anti-HIV drugs. Darunavir, in combination with ritonavir and other antiretroviral drugs, is indicated for the treatment of human immunodeficiency virus (HIV) infection in children aged 3 years and older and adults infected with HIV-1. FDA Label Darunavir, in combination with low-dose ritonavir, is indicated for the treatment of patients with human immunodeficiency virus (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 x 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 drugs. Darunavir (Krka dd) in combination with cobicistat is indicated for the treatment of HIV-1 infection in adults in combination with other antiretroviral drugs (see Section 4.2). Darunavir Krka dd 400 mg and 800 mg tablets are indicated for providing an appropriate dosing regimen for HIV-1 infection in adults and children aged 3 years and older and weighing at least 40 kg who are not currently receiving antiretroviral therapy (ART) (see Section 4.2). For patients who have previously received antiretroviral therapy (ART) and do not have darunavir resistance-associated mutations (DRV-RAMs), darunavir should be considered if their plasma HIV-1 RNA is < 100,000 copies/mL and their CD4+ cell count is ≥ 100 × 10⁶ cells/L. Genotyping results should guide the decision to initiate darunavir treatment in these previously ART-treated patients (see Sections 4.2, 4.3, 4.4, and 5.1). Darunavir Krka dd in 600 mg film-coated tablets, in combination with low-dose ritonavir, is indicated for the treatment of HIV-1 infection in patients with HIV infection in combination with other antiretroviral agents. Darunavir/Cecal double-dose tablets (600 mg) 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 large doses of treatment; and for the treatment of children aged 3 years and older with HIV-1 infected patients weighing at least 15 kg. When deciding to start darunavir in combination with low-dose ritonavir, the patient's previous 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.
Treatment of Human Immunodeficiency Virus (HIV-1) Infection

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Mechanism of Action
HIV-1 proteases are essential for the processing of viral precursor proteins and viral maturation, preparing for infection, and are therefore targets of HIV antiretroviral therapy. Protease inhibitors are part of highly active antiretroviral therapy (HAART) for HIV-infected patients. Studies have shown that HAART effectively suppresses the virus and significantly reduces 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 infected cells, preventing the formation of mature viral particles and thus stopping the spread of infection. Darunavir binds tightly to the primary chain of the amino acids (Asp-29 and Asp-30) at the active site of the protease, which may be one reason for its potency and efficacy against drug-resistant HIV-1 variants. Darunavir is known to bind to different sites on the enzyme: the active site lumen 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. Compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetic properties. Its chemical structure is related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum, potent inhibitor effective against all HIV-1 clinical isolates and exhibits extremely low cytotoxicity. We resolved the high-resolution crystal structures of the complexes of UIC-94017 with wild-type HIV-1 protease (PR) and mutant proteases PR (V82A) and PR (I84V), which are common in drug-resistant HIV. The refined resolution of these structures was 1.10–1.53 Å. The crystal structures of the PR and PR (I84V) ternary complexes with UIC-94017 showed that the inhibitor binds to the protease at two overlapping sites, while the inhibitor in the PR (V82A) complex has only one ordered binding site. In all three structures, UIC-94017 forms hydrogen bonds with the conserved backbone atoms Asp29 and Asp30 of the protease. These interactions are considered crucial to the activity of this compound against HIV isolates resistant to multiple protease inhibitors. Several other subtle differences exist in the interaction between the mutant and UIC-94017 compared to PR. PR (V82A) shows a difference in the backbone atomic position at residue 82 compared to the PR structure, which better accommodates the inhibitor. Finally, the 1.10 Å resolution structures of PR(V82A) and UIC-94017 showed an anomalous electron density distribution of catalytic aspartic acid residues, which is related to the reaction mechanism. [4] Darunavir is a second-generation HIV-1 protease inhibitor with a bis(tetrahydrofuran carbamate) backbone designed to enhance the activity of HIV-1 strains resistant to multiple protease inhibitors. [1,2] Mechanism: It binds to the active site of HIV-1 protease, blocking the cleavage of Gag-Pol polyprotein into mature proteins (p24, reverse transcriptase), thereby inhibiting viral assembly. [1,4] Approved use: It was approved by the FDA in 2006 for the treatment of HIV-1 infection in adults (in combination with ritonavir) and HIV-1 infection in children. Approved in 2014 for use in pediatric patients; for treatment-naïve or previously treated patients (including those resistant to protease inhibitors) [5] - Dosage forms: available in oral tablets, capsules and lipid nanoparticles (enhanced bioavailability and central nervous system delivery to target the latent HIV reservoir) [3,6]

C29H43N3O8S

593.73

593.277

C, 58.67; H, 7.30; N, 7.08; O, 21.56; S, 5.40

635728-49-3

Darunavir;206361-99-1

23725083

White to off-white solid powder

4.426

4

10

12

41

856

5

S(C1C([H])=C([H])C(=C([H])C=1[H])N([H])[H])(N(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])C([H])([H])[C@]([H])([C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(=O)O[C@@]1([H])C([H])([H])O[C@]2([H])[C@@]1([H])C([H])([H])C([H])([H])O2)O[H])(=O)=O.O([H])C([H])([H])C([H])([H])[H]

QWSHKNICRJHQCY-VBTXLZOXSA-N

InChI=1S/C27H37N3O7S.C2H6O/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;1-2-3/h3-11,18,22-26,31H,12-17,28H2,1-2H3,(H,29,32);3H,2H2,1H3/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;ethanol

Darunavir Ethanolate; TMC-114 Ethanolate; TMC114 Ethanolate; TMC 114 Ethanolate; UIC-94017 Ethanolate; UIC 94017 Ethanolate; UIC94017 Ethanolate; 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 (4.21 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.21 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.21 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.

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