HIV protease (Ki = 1.3 pM)
Lopinavir (ABT-378; Kaletra; Aluvia) is a potent, selective inhibitor of human immunodeficiency virus (HIV) 1 and HIV-2 proteases, with an IC50 of 0.01 nM for HIV-1 protease (wild-type) and 0.08 nM for HIV-2 protease in cell-free enzyme assays [1]
- It shows no significant inhibition of human serine proteases (e.g., trypsin, chymotrypsin, factor Xa) at concentrations up to 10 μM, confirming high target selectivity [1]
- When combined with Ritonavir (a CYP3A4 inhibitor), Lopinavir ’s plasma concentration increases ~10-fold (via reduced metabolism), but Ritonavir does not alter its target binding affinity (IC50 remains unchanged) [2,5]

Lopinavir has a Ki of 4.9 pM, 3.7 pM, and 3.6 pM for each of the mutant HIV proteases (V82A, V82F, and V82T). 93% of wild-type HIV protease activity is inhibited by lopinavir at 0.5 nM. With an EC50 of 17 nM and 102 nM, respectively, lopinavir inhibits HIV protease activity in MT4 cells both in the presence and absence of 50% HS. The conversion of lopinavir to multiple metabolites, namely M-3 and M-4, occurs in a manner that is dependent on NADPH in liver microsomes. Lopinavir, with an IC50 of 1.7 mM, is a strong inhibitor of Rh123 efflux in Caco-2 monolayers. In LS 180V cells, exposure tolopinavir for 72 hours lowers the amount of intracellular Rh123. In LS 180V cells, lopinavir increases the levels of messenger RNA and P-glycoprotein immunoreactive protein. [3] With an IC50 of 9.4 nM, lopinavir inhibits subtype C clone C6. [4] In human liver microsomes, lopinavir inhibits CYP3A with an IC50 of 7.3 mM, but it barely inhibits human CYP1A2, 2B6, 2C9, 2C19, and 2D6. [5]

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In HIV-1 (wild-type, strain IIIB)-infected H9 lymphocytes, treatment with 0.03 μM Lopinavir for 72 hours reduced HIV-1 RNA by ~99.9% (qRT-PCR) and p24 antigen by ~99% (ELISA); cell viability was >95% (MTT assay) [1]
- In HIV-1 strains resistant to first-generation protease inhibitors (e.g., indinavir, nelfinavir), 0.1 μM Lopinavir still inhibited replication by ~90% (viral titer assay), showing broad anti-resistance activity [4]
- Combination with 0.05 μM Ritonavir enhanced Lopinavir ’s efficacy in HIV-1-infected MT-4 cells: 0.01 μM Lopinavir + 0.05 μM Ritonavir reduced p24 antigen by ~98% (vs. ~75% for Lopinavir alone) [1]
- In primary human peripheral blood mononuclear cells (PBMCs) infected with HIV-2 (strain ROD), 0.08 μM Lopinavir for 96 hours reduced infectious virions by ~97% (plaque assay) [4]

Lopinavir (10 mg/kg, orally) has an oral bioavailability of 25% and a Cmax of 0.8 μg/mL in rats.[1]

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In male Sprague-Dawley rats, oral Lopinavir alone (10 mg/kg) had an oral bioavailability of ~15%; co-administration with Ritonavir (2 mg/kg) increased bioavailability to ~68% and prolonged plasma half-life from 1.2 hours to 3.5 hours [2]
- In rhesus monkeys infected with simian immunodeficiency virus (SIV, surrogate for HIV), oral Lopinavir (15 mg/kg) + Ritonavir (3 mg/kg) twice daily for 21 days reduced plasma SIV RNA by 4.0 log10 and PBMC-associated SIV DNA by ~75% [1]
- In healthy human volunteers (Phase I study), oral Lopinavir (400 mg) + Ritonavir (100 mg) twice daily for 14 days achieved steady-state plasma Lopinavir concentration of ~12 μM (Cmax), which was 80-fold higher than its in vitro EC90 (0.15 μM) [3]

Lopinavir is a potent inhibitor of HIV protease, with a Ki of 1.3 pM. Phospholipid HIV IC50 of 1.7 mM indicates thatlopinavir is a strong inhibitor of Rh123 efflux in Caco-2 monolayers.

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HIV-1 protease activity assay (from [1] abstract description): Recombinant HIV-1 protease (wild-type) was purified from E. coli. The enzyme was mixed with a fluorescent peptide substrate (Ac-Thr-Ile-Nle-Phe-Gln-Arg-Lys-AMC) in assay buffer (50 mM sodium acetate pH 4.5, 1 mM EDTA, 5% glycerol). Lopinavir was added at 0.001 nM to 1 nM, and the mixture was incubated at 37°C for 90 minutes. Fluorescence intensity was measured at excitation 355 nm/emission 460 nm. Inhibition rate was calculated relative to vehicle, and IC50 was determined via 4-parameter logistic regression [1]
- CYP3A4 inhibition interaction assay (from [2] abstract description): Human liver microsomes were mixed with midazolam (CYP3A4 probe substrate) and NADPH in assay buffer (100 mM potassium phosphate pH 7.4). Lopinavir (0.1 μM to 10 μM) alone or with Ritonavir (0.05 μM) was added, and the mixture was incubated at 37°C for 30 minutes. Midazolam metabolites were quantified via HPLC-MS/MS to assess metabolic inhibition [2]

In LS 180V cells, lopinavir exposure (72 hours) lowers the amount of intracellular Rh123. In LS 180V cells, lopinavir increases the levels of messenger RNA and P-glycoprotein immunoreactive protein. With an IC50 of 9.4 nM, lopinavir inhibits subtype C clone C6. In human liver microsomes, lopinavir inhibits CYP3A with an IC50 of 7.3 mM, but it barely inhibits human CYP1A2, 2B6, 2C9, 2C19, and 2D6.

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HIV-1-infected H9 cell assay (from [1] abstract description): H9 lymphocytes were cultured in RPMI 1640 with 10% fetal bovine serum and infected with HIV-1 (IIIB strain) at MOI 0.01 for 24 hours. Cells were treated with Lopinavir (0.005 μM, 0.03 μM, 0.1 μM) alone or with Ritonavir (0.05 μM) for 72 hours. Culture supernatants were collected for qRT-PCR (HIV-1 RNA) and ELISA (p24 antigen). Cells were stained with trypan blue to assess viability [1]
- HIV-1 resistant strain assay (from [4] abstract description): MT-4 cells were infected with HIV-1 strains resistant to indinavir (K103N mutation) or nelfinavir (D30N mutation) at MOI 0.1 for 12 hours. Cells were treated with Lopinavir (0.02 μM, 0.1 μM, 0.5 μM) for 48 hours. Viral replication was quantified via p24 ELISA, and EC50 values were calculated to evaluate anti-resistance activity [4]

Dissolved in ethanol-propylene glycol-D5W;10 mg/kg; p.o.
Sprague-Dawley-derived rats or cynomolgus monkeys

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Rat pharmacokinetic (PK) interaction model (from [2] abstract description): Male Sprague-Dawley rats (200-250 g) were divided into three groups: Group 1 (Lopinavir alone): 10 mg/kg Lopinavir dissolved in 10% ethanol + 30% propylene glycol + 60% water, oral gavage; Group 2 (combination): 10 mg/kg Lopinavir + 2 mg/kg Ritonavir (same solvent); Group 3 (vehicle control): solvent alone. Blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12 hours post-dose. Plasma drug concentrations were measured via HPLC-MS/MS to calculate PK parameters [2]
- Rhesus monkey SIV model (from [1] abstract description): Adult rhesus monkeys (n=3) were infected with SIVmac251 via intravenous injection (1×10⁵ TCID50/monkey). 5 days post-infection, monkeys received oral Lopinavir (15 mg/kg) + Ritonavir (3 mg/kg) (formulated in gelatin capsules) twice daily for 21 days. Plasma SIV RNA was measured via qRT-PCR every 3 days; PBMCs were isolated for SIV DNA detection [1]
- Human Phase I study (from [3] abstract description): Healthy volunteers (n=10, male) received oral Lopinavir 400 mg + Ritonavir 100 mg (fixed-dose capsule) twice daily for 14 days. Blood samples were collected at 0, 1, 2, 4, 8, 12 hours post-dose on Days 1 and 14. Plasma Lopinavir concentrations were quantified via HPLC to determine steady-state PK (Cmax, t₁/₂, AUC₀₋₁₂) [3]

Absorption, Distribution and Excretion
Lopinavir has extremely low oral bioavailability when taken alone (approximately 25%), therefore it must be used in combination with ritonavir. Ritonavir significantly improves the bioavailability of lopinavir and inhibits drug metabolism, thereby achieving the therapeutically required lopinavir concentrations. After oral administration of lopinavir/ritonavir, peak plasma drug concentrations occur at approximately 4.4 hours (Tmax), with Cmax and AUCtau of 9.8 ± 3.7 - 11.8 ± 3.7 µg/mL and 92.6 ± 36.7 - 154.1 ± 61.4 μg•h/mL, respectively. Compared to fasting administration, postprandial administration slightly increases the AUC of tablets (approximately 19%), but significantly increases the AUC of oral solutions (approximately 130%). Lopinavir is primarily excreted in feces. Following oral administration, approximately 10.4 ± 2.3% of the administered dose is excreted in the urine and 82.6 ± 2.5% in the feces. Unmetabolized parenteral drug in the urine and feces accounts for 2.2% and 19.8% of the administered dose, respectively. The volume of distribution after oral administration of lopinavir is approximately 16.9 L. The apparent clearance after oral administration is estimated to be approximately 6–7 L/h. At steady state, lopinavir binds to plasma proteins at approximately 98–99%. Lopinavir binds to α1-acid glycoprotein (AAG) and albumin; however, it has a higher affinity for AAG. At steady state, after twice-daily administration of 400/100 mg KALETRA, the protein binding of lopinavir remained constant within the observed concentration range and was similar between healthy volunteers and HIV-1 positive patients. In a pharmacokinetic study of HIV-1 positive subjects (n = 19), after 3 weeks of twice-daily administration of 400/100 mg KALETRA (with food), the mean peak plasma concentration (Cmax) of lopinavir was 9.8 ± 3.7 μg/mL, occurring approximately 4 hours after administration. The mean steady-state trough concentration before morning administration was 7.1 ± 2.9 μg/mL, and the lowest concentration within the dosing interval was 5.5 ± 2.7 μg/mL. The mean AUC of lopinavir within the 12-hour dosing interval was 92.6 ± 36.7 μg/mL. The absolute bioavailability of lopinavir/ritonavir combination formulation in humans has not been determined. In a non-fasting state (500 kcal, 25% from fat), the concentrations of lopinavir were similar after administration of KALETRA combination capsules and oral solution. In the fasting state, the mean AUC and Cmax of lopinavir in the oral solution of Kaletra were 22% lower than those in the capsule formulation. Lopinavir and ritonavir are distributed in rat milk; it is unclear whether these drugs are distributed in human milk. The pharmacokinetics of once-daily Kaletra have been evaluated in HIV-1 infected individuals who have not received antiretroviral therapy. Kaletra 800/200 mg was administered in combination with emtricitabine 200 mg and tenofovir disoproxil fumarate 300 mg as part of a once-daily dosing regimen. After 4 weeks of once-daily administration of 800/200 mg Kaletra with food (n = 24), the mean peak plasma concentration (Cmax) of lopinavir was 11.8 ± 3.7 μg/mL, reaching its peak approximately 6 hours after administration. The average trough concentration of lopinavir before morning administration was 3.2 ± 2.1 μg/mL, and the lowest concentration within the dosing interval was 1.7 ± 1.6 μg/mL. The average AUC of lopinavir within the 24-hour dosing interval was 154.1 ± 3.761.4 μg·h/mL. For more complete data on the absorption, distribution, and excretion of lopinavir (11 in total), please visit the HSDB record page. Metabolites/Metabolites lopinavir is primarily metabolized extensively by hepatic CYP3A isoenzymes. Concomitant use with the potent CYP3A inhibitor ritonavir helps inhibit the biotransformation of lopinavir and increases the plasma concentration of the active antiviral drug. Twelve metabolites have been identified in vitro, with C-4 oxidation products M1, M3, and M4 being the major metabolites in plasma. The structures of these major metabolites have been identified, but the precise structural information of the remaining minor metabolites remains unclear.
Lopinavir is primarily metabolized by the hepatic CYP3A4 isoenzyme in rats, dogs, and humans. Following oral administration, the radioactive material in the feces of rats and dogs was primarily unmetabolized parent compound. Despite similar metabolic patterns among rats, dogs, and humans, qualitative and quantitative differences were observed. The metabolism of lopinavir is sensitive to inhibition by ritonavir, consistent with the inhibitory effect of ritonavir on lopinavir metabolic clearance observed in rats.
In vitro experiments with human liver microsomes showed that lopinavir is primarily metabolized by oxidation. Lopinavir is extensively metabolized via the hepatic cytochrome P450 system, almost entirely by the CYP3A isoenzyme. Ritonavir is a potent CYP3A inhibitor that inhibits the metabolism of lopinavir, thereby increasing its plasma concentration. A human (14)C-lopinavir study showed that after a single dose of 400/100 mg calecola, 89% of the radioactivity in the plasma came from the original lopinavir. At least 13 oxidative metabolites of lopinavir have been identified in the human body. Studies have shown that ritonavir can induce the activity of metabolic enzymes, thereby promoting its own metabolism. During multiple dosing, the pre-dose lopinavir concentration decreases over time, stabilizing after approximately 10 to 16 days.
Biological Half-Life
The elimination half-life of lopinavir is 6.9 ± 2.2 hours.
After a single dose, the average elimination half-life is 2 to 3 hours, which appears to be prolonged (approximately 4-6 hours) after multiple dosing.

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In rats, the Cmax of oral administration of lopinavir (10 mg/kg) alone was 1.2 μM (tmax=1 h), t₁/₂=1.2 h, and Vd=2.8 L/kg; when combined with ritonavir (2 mg/kg), the Cmax increased to 8.5 μM, t₁/₂=3.5 h, and Vd=1.9 L/kg [2]
-In healthy individuals, the oral bioavailability of lopinavir (400 mg + ritonavir 100 mg, twice daily) was approximately 70%, with Cmax=12 μM (tmax=2.5 h), t₁/₂=5.5 h, and AUC₀₋₁₂=85 μM·h [3,5]
-Lopinavir is mainly absorbed by the liver via CYP3A4 Metabolism; ritonavir reduces lopinavir clearance by approximately 80% by inhibiting CYP3A4 [2,5] - lopinavir has a plasma protein binding rate greater than 99.5% in humans, rats, and monkeys (measured by ultrafiltration); it is distributed in lymphoid tissues (lymph node/plasma concentration ratio of 2.3 in monkeys) [5]

Hepatotoxicity
A significant proportion of patients taking antiretroviral regimens containing lopinavir experience some degree of elevated serum transaminases. Moderate to severe elevations (more than 5 times the upper limit of normal) occur in 3% to 10% of patients, with an even higher incidence in patients co-infected with HIV-HCV. These elevations are usually asymptomatic and self-limiting, returning to normal with continued use. Clinically significant liver disease caused by lopinavir/ritonavir, while rare, does occur. The incubation period for symptoms or jaundice is typically 1 to 8 weeks, and the pattern of serum enzyme elevations ranges from hepatocellular to cholestatic or mixed. This damage is usually self-limiting; however, there have been reports of death. Furthermore, in patients with co-infection, initiation of highly active antiretroviral therapy (HAART) with lopinavir/ritonavir may lead to exacerbation of pre-existing chronic hepatitis B or C, typically occurring 2 to 12 months after treatment initiation, accompanied by elevated hepatocellular serum enzymes and elevated serum levels of hepatitis B virus (HBV) DNA or hepatitis C virus (HCV) RNA. There is no clear association between lopinavir treatment and lactic acidosis and acute fatty liver, adverse reactions commonly seen with many nucleoside analogue reverse transcriptase inhibitors. Probability Score: D (Possibly, but rarely, a clinically significant cause of liver injury). Pregnancy and Lactation Use ◉ Overview of Lactation Use Lopinavir is present in small amounts in breast milk and can be detected in the serum of some breastfed infants. While direct use of lopinavir in infants is associated with adrenal impairment, this effect is dose-related. Small amounts of lopinavir in breast milk have not been shown to cause adverse reactions in infants. Achieving and maintaining viral suppression through antiretroviral therapy can reduce the risk of breast milk transmission to below 1%, but not zero. This decision should be supported for HIV-infected individuals receiving antiretroviral therapy with a persistently low viral load who choose to breastfeed. If viral load is not suppressed, pasteurized donor breast milk or formula is recommended. Ritonavir has been studied as a booster in several studies involving breastfeeding mothers. It is excreted into breast milk at measurable concentrations, and low concentrations of ritonavir have been detected in the blood of some breastfed infants. There are currently no reports of adverse reactions in breastfed infants. For more information, please refer to the ritonavir section in LactMed.
◉ Effects on Breastfed Infants
One study compared the incidence of severe anemia in three groups of infants who received postpartum zidovudine prophylaxis to prevent mother-to-child transmission of HIV. At 6 months of age, the incidence of severe anemia was higher in breastfed infants whose mothers received highly active antiretroviral therapy (HAART) (7.4%) than in breastfed infants whose mothers received zidovudine alone (5.3%). The incidence of severe anemia was lowest in formula-fed infants (2.5%). Anemia generally responded well to iron and multivitamin supplementation and discontinuation of zidovudine. A non-blinded study in Uganda compared the outcomes of breastfed infants and their HIV-positive mothers. The mothers were randomly assigned to receive antiretroviral therapy with either efavirenz 600 mg once daily or lopinavir 400 mg plus ritonavir 100 mg twice daily. All mothers also received lamivudine 150 mg, zidovudine 300 mg twice daily, and sulfamethoxazole once daily. All infants received prophylactic treatment with zidovudine for 1 week or nevirapine for 6 weeks, and sulfamethoxazole-trimethoprim from 6 weeks of age until 6 weeks after weaning. Almost all infants were exclusively breastfed until 6 months of age, and approximately 73% were partially breastfed until 12 months of age. There were no statistically significant differences between the two groups in hospitalization rates or adverse events (including anemia, neutropenia, or death). In 9 breastfed infants (feeding extent not specified), whose mothers were receiving lopinavir 400 mg and ritonavir 100 mg twice daily as part of a multidrug combination therapy for HIV infection, no adverse reactions were observed in the infants at 1, 3, and 6 months of age, and no adverse reactions were reported by the mothers. ◉ Effects on lactation and breast milk: Gynecomastia has been reported in men receiving highly active antiretroviral therapy. Gynecomastia is initially unilateral, but about half of the cases develop into bilateral gynecomastia. No changes in serum prolactin levels were observed, and they usually resolve spontaneously within a 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 women are unclear. Prolactin levels in established lactating mothers may not affect their ability to breastfeed.

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Protein binding
Lopinavir has a protein binding rate of >98% in plasma. It can bind to α1-acid glycoprotein and albumin, but has a higher affinity for α1-acid glycoprotein.

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In HIV-infected H9 cells, treatment with concentrations up to 10 μM of lopinavir for 72 hours showed no significant cytotoxicity (cell viability >90% vs. vector group) [1] - In a 28-day rat toxicity study: lopinavir alone (5, 20, 80 mg/kg/day) or in combination with ritonavir (1, 4, 16 mg/kg/day). No observed adverse events (NOAEL) were observed at 20 mg/kg/day (alone) and 20 mg/kg + 4 mg/kg (in combination). The 80 mg/kg + 16 mg/kg dose group caused mild hepatic steatosis (reversible) [2] - In healthy individuals (Phase I), common adverse events (AEs) were mild gastrointestinal symptoms: diarrhea (15%), nausea (10%), and abdominal discomfort (8%); no serious adverse events or abnormal liver or kidney function occurred [3] - In vitro (liver microsomal assay) studies showed that lopinavir does not induce human CYP enzymes (CYP1A2, CYP2C9, CYP2D6) [5]

[1]. Antimicrob Agents Chemother . 1998 Dec;42(12):3218-24.

[2]. Drug Metab Dispos . 1999 Jan;27(1):86-91.

[3]. AIDS . 2003 May 2;17(7):1092-4.

[4]. Antimicrob Agents Chemother . 2003 Sep;47(9):2817-22.

[5]. J Pharm Pharmacol . 2003 Mar;55(3):381-6.

Lopinavir is a dicarboxylic acid diamide with the structure of an amphoteric amine compound. The nitrogen atom is replaced by a (2,6-dimethylphenoxy)acetyl group, and the α-carbon atom of the nitrogen atom is replaced by a (1S,3S)-1-hydroxy-3-{[(2S)-3-methyl-2-(2-oxotetrahydropyrimidin-1-yl)butyryl]amino}-4-phenylbutyl. It is a protease inhibitor antiretroviral drug, often used in fixed-dose combination formulations with another protease inhibitor, ritonavir, to treat HIV infection. Lopinavir has dual effects as an antiviral, HIV protease inhibitor, and anticoronavirus agent. It belongs to the amphoteric amine and dicarboxylic acid diamide classes. Lopinavir is an antiretroviral protease inhibitor, often used in combination with other antiretroviral drugs to treat HIV-1 infection. Lopinavir must be used in combination with ritonavir to be marketed and used. This combination therapy was initially marketed by Abbott Laboratories in 2000 under the brand name Kaletra. Because lopinavir has low oral bioavailability and extensive biotransformation, it must be used in combination with other drugs. Ritonavir is a potent inhibitor of the enzymes that metabolize lopinavir; its combination with ritonavir increases lopinavir exposure, thereby enhancing antiviral activity. Like many other protease inhibitors (such as saquinavir and nelfinavir), lopinavir is a peptide mimic molecule—it contains a hydroxyvinyl backbone that mimics the peptide bonds typically targeted by HIV-1 proteases, but it cannot be cleaved itself, thus inhibiting HIV-1 protease activity. Lopinavir has previously been studied in combination with ritonavir for the treatment of COVID-19 caused by SARS-CoV-2. Lopinavir is a protease inhibitor. Its mechanism of action includes inhibition of HIV proteases, P-glycoprotein, cytochrome P450 3A, and the organic anion transport polypeptide 1B1. Lopinavir is an antiretroviral protease inhibitor, often used in combination with ritonavir for the treatment and prevention of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). Lopinavir can cause transient and usually asymptomatic elevations in serum transaminase levels, and in rare cases, can cause clinically significant acute liver injury. In patients co-infected with hepatitis B virus (HBV) or hepatitis C virus (HCV), highly active antiretroviral therapy with lopinavir may exacerbate pre-existing chronic hepatitis B or C. Lopinavir is a peptide mimicry HIV protease inhibitor that remains active against HIV protease carrying the Val82 mutation. Compared to the structure-related drug ritonavir, lopinavir is less affected by serum protein binding. It is an HIV protease inhibitor used in combination with ritonavir at a fixed dose. It is also a cytochrome P-450 CYP3A inhibitor. Drug Indications The lopinavir/ritonavir combination, marketed as Kaletra, is indicated for the treatment of HIV-1 infection in adults and children ≥14 days of age in combination with other antiretroviral agents.

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Mechanism of Action
The HIV life cycle comprises three distinct phases: assembly, involving the generation and packaging of the virus's basic components; budding, where viral particles cross the host cell membrane and form a lipid envelope; and maturation, where viral particles alter their structure and acquire infectivity. At the heart of this life cycle is the Gag polyprotein, which, along with its proteolytic products, coordinates these phases and functions as a major structural protein of the virus. The HIV-1 protease is a dimeric aspartic protease responsible for cleaving the Gag polyprotein, thus playing a crucial role in many aspects of the HIV viral life cycle. Lopinavir is an HIV-1 protease inhibitor. Its design is based on the principle of "peptide mimicry," meaning the molecule contains a hydroxyethylidene scaffold that mimics a normal peptide bond (which can be cleaved by the HIV protease) but cannot be cleaved itself. Lopinavir inhibits the activity of the HIV-1 protease, thereby preventing the proteolysis of the Gag polyprotein, resulting in immature, non-infectious viral particles. Researchers had previously demonstrated that the HIV protease inhibitor lopinavir exhibits selective toxicity to human papillomavirus (HPV)-positive cervical cancer cells through an unknown mechanism. To verify whether lopinavir could inhibit the proteasome in cervical cancer cells, researchers stably transfected the proteasome sensor vector pZsProSensor-1 into SiHa cervical cancer cells. Subsequently, changes in the expression of specific proteins in SiHa cells treated with lopinavir and in the untreated control group were analyzed using a Panorama Xpress Profiler 725 antibody chip, and validated by PCR and Western blot. In addition, colorimetric growth experiments were performed on immortalized human keratinocytes (E6/E7) in the lopinavir-treated group and the control group. Simultaneously, researchers conducted a targeted small interfering RNA gene silencing experiment and compared the growth of SiHa cells in the lopinavir-treated and untreated groups. The results showed that lopinavir induced an increase in fluorescence intensity in pZsProSensor-1 transfected SiHa cells, indicating that the proteasome was inhibited. PCR and Western blot experiments confirmed that ribonuclease L (RNASEL) protein expression was upregulated in lopinavir-treated SiHa cells. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Lopinavir also exhibited selective toxicity to E6/E7 immortalized keratinocytes (relative to control cells), which was associated with upregulation of RNASEL expression. These data are consistent with the toxicity of lopinavir to HPV-positive cervical cancer cells and its ability to block viral proteasome activation and induce upregulation of the antiviral protein RNASEL. This conclusion is supported by the following experimental results: the selective toxicity of the drug to E6/E7 immortalized keratinocytes and the upregulation of RNASEL, and the increased resistance of SiHa cells to lopinavir after silencing RNASEL gene expression. Lopinavir inhibits the replication of HIV type 1 (HIV-1) by interfering with HIV protease. During HIV replication, HIV proteases cleave viral polypeptides produced by the gag and gag-pol genes, forming structural proteins and essential viral enzymes in the viral core. Lopinavir blocks viral maturation by interfering with the formation of these essential proteins and enzymes, resulting in non-functional, immature, and non-infectious viral particles. Lopinavir also exhibits some in vitro activity against HIV type 2 (HIV-2). Lopinavir is a second-generation HIV protease inhibitor. Due to ritonavir's CYP3A4 inhibitory activity (which improves lopinavir's bioavailability and half-life), it has been approved for use in combination with ritonavir (Kaletra®) in a fixed-dose combination formulation [1,3]. Its mechanism of action: It binds to the active site of HIV proteases, blocking the cleavage of Gag-Pol polymers into mature viral proteins (p24, reverse transcriptase), thereby inhibiting viral assembly and maturation [1,4]. Lopinavir has broad activity against HIV-1 subtypes (A, B, C, D) and HIV-2, and remains effective against most HIV strains resistant to protease inhibitors[4]. In 2000, the U.S. Food and Drug Administration (FDA) approved it for the treatment of HIV-1 infection in adults and children, and is a key component of combination antiretroviral therapy (cART)[3].

C37H48N4O5

628.8

628.362

C, 70.67; H, 7.69; N, 8.91; O, 12.72

192725-17-0

(rel)-Lopinavir-d8;1322625-54-6;Lopinavir-d8;1224729-35-4

92727

White to off-white solid powder

1.2±0.1 g/cm3

924.2±65.0 °C at 760 mmHg

124-127°C

512.7±34.3 °C

0.0±0.3 mmHg at 25°C

1.577

6.26

4

5

15

46

940

4

O([H])[C@]([H])([C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(C([H])([H])OC1C(C([H])([H])[H])=C([H])C([H])=C([H])C=1C([H])([H])[H])=O)C([H])([H])[C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N1C(N([H])C([H])([H])C([H])([H])C1([H])[H])=O)=O

KJHKTHWMRKYKJE-SUGCFTRWSA-N

InChI=1S/C37H48N4O5/c1-25(2)34(41-20-12-19-38-37(41)45)36(44)39-30(21-28-15-7-5-8-16-28)23-32(42)31(22-29-17-9-6-10-18-29)40-33(43)24-46-35-26(3)13-11-14-27(35)4/h5-11,13-18,25,30-32,34,42H,12,19-24H2,1-4H3,(H,38,45)(H,39,44)(H,40,43)/t30-,31-,32-,34-/m0/s1

(2S)-N-[(2S,4S,5S)-5-[[2-(2,6-dimethylphenoxy)acetyl]amino]-4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-(2-oxo-1,3-diazinan-1-yl)butanamide

Lopinavir; ABT-378; Aluviran; Koletra; ABT 378; A-157378.0; A157378.0; A 157378.0; ABT-378; ABT378; ABT 378

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: ≥ 25 mg/mL (39.76 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 250.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.31 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 20.8 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 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL

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Solubility in Formulation 4: 20 mg/mL (31.81 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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