EGFR 10.8 nM (IC50) ErbB2 9.2 nM (IC50)

In BT474 and HN5 cells, lapatinib (GW2016; 0.03-10 µM; 6 hours) therapy suppresses EGFR and ErbB-2 receptor autophosphorylation in a dose-dependent manner. GW2016 suppressed AKT's serine 473 phosphorylation in a dose-dependent manner [1]. The administration of lapatinib (GW2016; 72 hours; HN5, A-43, BT474, N87, and CaLu-3 cells) selectively inhibits the growth of human tumor cell lines[1]. G1 arrest is induced by lapatinib (GW2016; 1- 10 µM; 72 hours; HN5 cells) treatment[1].

Treatment with lapatinib (GW2016; 30-100 mg/kg; oral administration; twice daily; for 21 days; CD-1 nude female mice) significantly reduces the growth of HN5 tumor xenografts at doses of 30 and 100 mg/kg, completely inhibiting tumor growth at the higher dose[1]. Lapatinib (~100 mg/kg) given orally twice a day significantly and dose-dependently inhibits the growth of HN5 and BT474 xenografts.[1]

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The combination of lapatinib with radiation therapy suppressed the growth of MBT-2 xenograft tumors in mice [2]
In mice with tumor xenografts, a daily dose of lapatinib (200 mg/kg/day) for seven days combined with radiation on the fourth day suppressed tumor growth to a greater degree than radiation alone. The outcomes of lapatinib treatment of tumor xenografts in this animal model showed that a daily dose of lapatinib (oral, 200 mg/kg/day) for seven days, combined with radiation on the fourth day caused a significant suppression in the growth of xenografts tumors compared with irradiation alone (Figure 6A). However, oral lapatinib treatment alone had minimal effect. The results suggested that an oral dose of lapatinib increased the radiation-mediated suppression of xenografts tumors by about 60%. The results of immunohistochemistry for expression of HER-2 and EGFR in tumors recovered from mice at the end of treatment protocol of seven days showed the involvement of radiation in enhancing the levels of EGFR and HER-2 (Figure 6B). However, lapatinib, in combination with radiation therapy, suppressed the radiation-mediated activation of EGFR and HER-2 in xenograft tumors. The outcomes of this in vivo experiment indicated that lapatinib induced radiosensitization by inhibiting the radiation-mediated expression of EGFR and HER-2, in addition to facilitating DNA damage. [2]

The process of measuring the inhibition of phosphorylation of a peptide substrate yields the IC50 values for inhibition of enzyme activity. The EGFR and ErbB2 intracellular kinase domains are isolated using a baculovirus expression system. In round-bottomed polystyrene 96-well plates, EGFR and ErbB2 reactions are carried out with a final volume of 45 μL. The reaction mixtures consist of the following components: 50 μM Peptide A [Biotin-(amino hexonoic acid)-EEEEYFELVAKKK-CONH2], 1 mM dithiothreitol, 2 mM MnCl2, 10 μM ATP, 1 μCi of [γ33P] ATP/reaction, and 1 μL of DMSO containing serial dilutions of Lapatinib starting at 10 μM. The indicated purified type-1 receptor intracellular domain is added to start the reaction. One pmol of added enzyme is used for each reaction (20 nM). After 10 minutes at 23°C, 45 μL of 0.5% phosphoric acid in water is added to stop the reaction. The 75 μL of the finished reaction mix is put onto phosphocellulose filter plates. The plates undergo three rounds of filtering and washing with 200 μL of 0.5% phosphoric acid. Each well receives 50 μL of the scintillation cocktail, and the assay is measured using a Packard Topcount. 10-point dose-response curves are used to calculate IC50 values.

Western Blot Analysis[1]
Cell Types: BT474 and HN5 cells
Tested Concentrations: 0.03 µM, 0.1 µM, 0.3 µM, 1 µM, 3 µM, or 10 µM
Incubation Duration: 6 hrs (hours)
Experimental Results: Inhibited receptor autophosphorylation of EGFR and ErbB-2 in a dose-responsive manner. Phosphorylation of serine 473 of AKT was also inhibited in a dose-dependent manner. Cell Proliferation Assay[1]
Cell Types: HN5, A-43, BT474, N87, and CaLu-3 cells
Tested Concentrations:
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibited the growth of tumor cells overexpressing EGFR or ErbB-2.

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Cell Cycle Analysis[1]
Cell Types: HN5 cells
Tested Concentrations: 1 µM, or 10 µM
Incubation Duration: 72 hrs (hours)
Experimental Results: Resulted in induction of G1 arrest.

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For 72 hours, cells are exposed to different lapatinib concentrations. Methylene blue staining is used to estimate the relative number of cells. A Spectra microplate reader is used to measure the absorbance at 620 nm. Propidium iodide staining, antibody detection of incorporated BrdUrd, and propidium iodide staining are used to analyze cell death and the cell cycle.

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Clonogenic assay (colony formation assay) [2]
To test the effects of lapatinib and irradiation on colony formation, cells were seeded using six-well plates and a cell density of 1×105 cells/well. The cells were exposed to different radiation doses, but received pretreatment with lapatinib (200–1,000 nM) for 30 min, with the control cells treated with dimethyl sulfoxide (DMSO). After pre-treatment with lapatinib, and following irradiation, the cells were cultured for a further week. Counting of the cell colonies was done using a light microscope (×100 magnification), and the colonies were defined as a group of 50 cells or more.

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Cell cycle analysis [2]
The cell cycle distribution was done by flow cytometry analysis. Propidium iodide (PI) staining for DNA in cells was analyzed. For the protocol, 106 cells/ml were exposed to lapatinib and irradiation as previously described and were collected after centrifugation. The cells were stained with PI (15 μg/ml) in PBS with 5 μg/ml DNase-free RNase and Tween-20 (0.5%). The samples were analyzed using an Attune™ NxT Acoustic Focusing Cytometer.

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Immunofluorescence microscopic studies [2]
The MBT-2 cells were transferred onto coverslips pre-coated with poly-lysine for 12 h to allow the cells to attach to the surface. The cells were exposed to a radiation dose of 2.5 Gy either alone, or in combination with 100 nM of lapatinib . The cells were then incubated for 45 min and were then washed three times with ice-cold PBS, then treated for 30 min with a 4% solution of formaldehyde in PBS for fixation, followed by incubation in 0.5% Triton X-100/PBS for 60 min, 5% bovine serum albumin (BSA) for 60 min, and a final incubation for 2 h with fluorescein isothiocyanate (FITC)-conjugated anti-phospho-Histone γ-H2AX antibody (1: 1500). The cells were washed with PBS and mounted in Vectashield mounting medium containing diamidino-2-phenylindole. A Zeiss LSM 8 microscope was used to examine the γ-H2AX nuclei at high power, and a mean of at least 120 nuclei was counted. The mean of the γ-H2AX foci/nuclei indicated the number of DNA double-strand breaks.

Animal/Disease Models: CD-1 nude female mice (4-6 weeks old) with HN5 cells[1]
Doses: 30 mg/kg, 100 mg/kg
Route of Administration: Oral administration; twice (two times) daily; for 21 days
Experimental Results: Inhibited tumor xenograft growth of the HN5 cells in a dose-responsive manner.

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The C3H/HEN mice were inoculated with a subcutaneous injection of a suspension of MBT-2 cells (100 μl) (1×107 cells/100 μl) into the right flank of the mice on day 1. After one week, the tumor size was measured using vernier calipers, and the volume was calculated. A mean volume of 162 mm3 was regarded as a criterion for tumor establishment. After successful establishment of tumor, the mice were divided into four groups: Group 1, the control group (vehicle treated with 0.5% methylcellulose and 0.1% Tween-80); Group 2, lapatinib -treated (200 mg/kg/day); Group 3, vehicle and irradiation (15 Gy) on day 4; Group 4, lapatinib -treated (200 mg/kg/day) and irradiated (15 Gy) on the 4th day. The body weight of all the mice was recorded every week. Positron emission tomography (PET) and computed tomography (CT) scans were taken (PET/CT) by intravenous injection of the animals with 14 MBq (378 Ci) of fludeoxyglucose (FDG) in saline via the tail vein. [2]

Absorption, Distribution and Excretion
Absorption of lapatinib after oral administration is incomplete and varies considerably among individuals. Lapatinib is primarily metabolized via CYP3A4 and CYP3A5, with minor involvement from CYP2C19 and CYP2C8, generating various oxidative metabolites. However, the content of these metabolites in feces does not exceed 14% of the drug dose, and the concentration of lapatinib in plasma is 10%. In mice, rats, and dogs, the primary route of excretion of drug-related substances after a single oral administration of (14)C-lapatinib is feces, with very little excretion in urine. Most of the dose is cleared within 48 hours of administration. Lapatinib is primarily eliminated via metabolism via CYP3A4/5, with negligible renal excretion (<2%). The median recovery of parent lapatinib in feces after an oral dose was 27% (range 3% to 67%). Co-administration with food increases systemic exposure to lapatinib. When taken with a low-fat meal (5% fat, 500 calories), lapatinib's AUC increased by approximately 3-fold and 4-fold (Cmax increased by approximately 2.5-fold and 3-fold, respectively); when taken with a high-fat meal (50% fat, 1000 calories), lapatinib's AUC increased by approximately 2.5-fold and 3-fold, respectively. Lapatinib exhibits high binding rates (>99%) to albumin and α1-acid glycoprotein. In vitro studies have shown that lapatinib is a substrate for the transporters of breast cancer resistance protein (BCRP, ABCG2) and P-glycoprotein (P-gp, ABCB1). In vitro studies have shown that lapatinib inhibits P-gp, BCRP, and the hepatic uptake transporter OATP 1B1 at clinically relevant concentrations. For more complete data on the absorption, distribution, and excretion of lapatinib (7 studies), please visit the HSDB records page.

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Metabolism/Metabolites
Lapatinib is primarily metabolized via CYP3A4 and CYP3A5, with minor involvement from CYP2C19 and CYP2C8, generating various oxidative metabolites. The recovery rate of any single metabolite in feces does not exceed 14%, and its concentration in plasma does not exceed 10%.
Lapatinib is an oral breast cancer drug, recently reported as a mechanism-based cytochrome P450 (P450) 3A4 inactivator and a specific inhibitor. Hepatotoxicity. Studies have shown that the formation of active quinone imine metabolites is associated with mechanism-based inactivation (MBI) and/or hepatotoxicity. We investigated the MBI mechanism of lapatinib on P450 3A4. Liquid chromatography-mass spectrometry analysis showed that no peaks corresponding to irreversible modification appeared in P450 3A4 after lapatinib incubation. The enzyme activity inactivated by lapatinib was completely restored upon the addition of potassium ferricyanide. These results indicate that the MBI mechanism of lapatinib is quasi-irreversible and mediated by the formation of a metabolic intermediate complex (MI complex). This finding is confirmed by an increase in characteristic Soret uptake at approximately 455 nm. Two amine oxidation products generated from lapatinib metabolism via P450 3A4 were characterized: N-hydroxylapatinib (M3) and the oxime form of N-dealkylated lapatinib (M2), suggesting that nitroso or other related intermediates generated from M3 are involved in the formation of the MI complex. In contrast, P450 3A5 is significantly less sensitive to lapatinib MBI via the MI complex formation pathway than P450 3A4. Furthermore, P450 3A5's ability to generate M3 is also significantly lower than that of 3A4, consistent with N-hydroxylation being the initial step in the MI complex formation pathway. In summary, our results indicate that the primary mechanism of lapatinib's MBI effect on P450 3A4 is not the irreversible modification of quinone imine metabolites, but rather the formation of a quasi-irreversible MI complex mediated by the oxidation of lapatinib's secondary amine group. Lapatinib is extensively metabolized in the human body, generating various oxidation products as well as N- and O-dealkylated products. In vitro studies using human hepatocytes and microsomes show that lapatinib is mainly metabolized by CYP3A4 and CYP3A5, with a smaller contribution from CYP2C8. Other studies suggest that CYP1A2, 2D6, 2C9, and 2C19 may also be involved in metabolism, but to a lesser extent. The most important metabolites are carboxylic acid GW42393 and O-dealkylated phenol GW690006. The N-oxidation of secondary fatty amines produces a series of approximately eight minor metabolites. Compared to the parent drug, GW690006 showed roughly the same inhibitory effect on ErbB1-dependent tumor cell growth in vitro, but its inhibitory effect on ErbB2-dependent tumor cells was reduced by approximately 100-fold. GW342393 showed approximately 40-fold reduced inhibitory effects on both ErbB1 and ErbB2-dependent tumor cells compared to the parent drug. They are unlikely to contribute to the bioactivity of lapatinib. Lapatinib is an oral tyrosine kinase inhibitor used to treat breast cancer and has been reported to cause specific hepatotoxicity. Recently, it was discovered that lapatinib forms a metabolite-inhibitor complex (MIC) with CYP3A4 by forming an alkylnitroso intermediate. Because CYP3A5 is highly polymorphic compared to CYP3A4 and can oxidize lapatinib, we investigated the interaction between lapatinib and CYP3A5. Using testosterone as a probe substrate, lapatinib inactivated CYP3A5 in a time-, concentration-, and NADPH-dependent manner, with K_I and k_inact values of 0.0376 mM and 0.0226 min^-1, respectively. However, similar results were not obtained when midazolam was used as a probe substrate, indicating that lapatinib has site-specific inactivation of CYP3A5. Poor recovery of CYP3A5 activity after dialysis and the absence of a Soret peak confirmed that lapatinib does not form a minimum inhibitory concentration (MIC) with CYP3A5. Reduced CO differential spectroscopy further indicated that most of the active metabolites of lapatinib are covalently bound to the apolipoproteins of CYP3A5. Capture of the active metabolites of lapatinib generated from CYP3A5 using GSH confirmed the formation of a quinone imine-GSH adduct derived from the O-dealkylated metabolite of lapatinib. Computer simulations of docking studies support the preferential formation of O-dealkylated metabolites of lapatinib by CYP3A5, rather than the N-hydroxylation reaction primarily catalyzed by CYP3A4. In summary, lapatinib appears to be a mechanistic inhibitor of CYP3A5 through adduct with quinone imine metabolites. Following a single oral administration of (14)C-lapatinib, the metabolism of lapatinib in plasma and excreta in rats (10 mg/kg), dogs (10 mg/kg), mice (30 mg/kg), and humans (250 mg) was quantitatively and qualitatively assessed. Overall, (14)C-lapatinib is primarily metabolized, secreted into bile, and ultimately excreted in feces. Urine samples were not analyzed in non-clinical and clinical metabolic studies due to the low proportion of dose excreted in urine. In plasma, (14)C-lapatinib was the most abundant single component across all species. Male rats metabolized lapatinib more extensively than female rats, but the metabolic profiles were similar. In dogs and humans, 14C-lapatinib was the only quantitatively detectable peak. In humans, lapatinib accounts for only about half of the plasma radioactivity. The remaining radioactivity is attributed to at least eight metabolites detected by liquid chromatography-mass spectrometry (LC-MS), but these metabolites are present in amounts below the radiochemical limit of quantitation (approximately 5% of the total mixed plasma radioactivity). These metabolites are attributed to the N-oxidation cascade, which has been observed in vitro and in rats and mice. In mice and rats, only a few metabolites are quantified in plasma by radiochemical detection, but all metabolites have been characterized by mass spectrometry. Therefore, no unique circulating metabolites were observed in humans.

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Biological Half-Life
Single-dose terminal half-life: 14.2 hours; Effective multiple-dose half-life: 24 hours
At clinical doses, the terminal half-life after a single dose is 14.2 hours; drug accumulation after repeated dosing indicates an effective half-life of 24 hours. In a mass balance study, six healthy volunteers were given a single dose of 250 mg of (14)C-labeled lapatinib. The results showed that serum concentrations of the radiolabeled substances, representing the parent drug and its metabolites, peaked 4 hours after administration and decreased over a median half-life of 6 hours. Plasma concentrations of lapatinib decreased over a half-life of 14 hours.

Toxicity Summary
Identification and Uses: Lapatinib is a yellow solid, formulated as film-coated tablets. Lapatinib is an antineoplastic drug that inhibits human epidermal growth factor receptor type 2 (HER2/ERBB2) and epidermal growth factor receptor (HER1/EGFR/ERBB1) tyrosine kinases. It is used in combination with capecitabine to treat patients with HER2-overexpressing advanced or metastatic breast cancer who have previously received anthracyclines, taxanes, or trastuzumab. It is also used in combination with letrozole to treat postmenopausal women with hormone receptor-positive, HER2-overexpressing breast cancer who require hormone therapy. Human Exposure and Toxicity: Asymptomatic and symptomatic overdose cases have been reported. The daily dose range is 2,500 to 9,000 mg for 1 to 17 days. Observed symptoms include lapatinib-related events, with some cases also experiencing scalp pain, sinus tachycardia (with a normal ECG), and/or mucosal inflammation. Hepatotoxicity, manifested as elevated serum transaminase and bilirubin levels, has been observed at therapeutic doses in clinical trials and post-marketing experience with lapatinib. Hepatotoxicity can be severe, and deaths have been reported. The cause of death is unclear. Hepatotoxicity may occur within days to months after initiation of treatment. Lapatinib should be avoided in pregnant women. Although there are currently no adequate and well-controlled studies in pregnant women, lapatinib has been associated with adverse reproductive effects in animal studies. If used during pregnancy, patients should be informed of the potential fetal risks. Animal studies: A two-year mouse study found no evidence of carcinogenicity, but increased mortality associated with skin toxicity was observed in male mice at doses of 150 and 300 mg/kg/day and in female mice at a dose of 300 mg/kg/day. A two-year rat carcinogenicity study found increased mortality, associated with skin toxicity, in male rats at a dose of 500 mg/kg/day and in female rats at a dose of 300 mg/kg/day. Female rats at doses of 60 mg/kg/day and 180 mg/kg/day, respectively, developed renal infarction and renal papillary necrosis. The incidence of benign hemangiomas in the mesenteric lymph nodes was increased in male rats at doses of 120 mg/kg/day and in female rats at doses of 180 mg/kg/day, but remained within the background range. The clinical significance of these findings in humans is unclear. Lapatinib at doses up to 120 mg/kg/day in female rats and up to 180 mg/kg/day in male rats did not affect gonadal function, mating, or fertility in either male or female rats. Studies in pregnant rats and rabbits showed that lapatinib had no teratogenic effects. However, in rats, some minor malformations (left umbilical artery, neck ribs, and premature ossification) occurred at maternally toxic doses of 120 mg/kg/day. In rabbits, lapatinib was associated with maternal toxicity at doses of 60 and 120 mg/kg/day, and caused abortion at a dose of 120 mg/kg/day. At maternally toxic doses, decreased fetal weight, reduced live birth count, and mild skeletal malformations were observed. Lapatinib did not show chromosomal breakage or mutagenicity in a series of assays, including the Chinese hamster chromosomal aberration assay, Ames assay, human lymphocyte chromosomal aberration assay, and in vivo rat bone marrow chromosomal aberration assay.
Hepatotoxicity
Elevated serum transaminase levels are common during lapatinib treatment, occurring in up to half of patients. 2% to 6% of patients experience transaminase levels exceeding 5 times the upper limit of normal (ULN), but this is usually transient and asymptomatic. Dose adjustments or temporary discontinuation due to abnormal liver function are rare.
Since the clinical introduction of lapatinib, several cases of clinically significant acute liver injury have been associated with lapatinib. The clinical features of liver injury are not well-defined, but it typically appears within 1 to 3 months of starting lapatinib treatment, with serum enzyme elevations usually presenting as hepatocellular or mixed (Case 1). The U.S. Food and Drug Administration (FDA) has received sufficient reports of liver injury to classify lapatinib as a potentially fatal hepatotoxic drug. The incidence of severe liver injury is estimated at 0.2%, but may be higher. Immune hypersensitivity and autoimmune features are uncommon, but genetic studies have shown that lapatinib hepatotoxicity is associated with specific HLA alleles. Most cases are self-limiting, but there have been several reports of acute liver failure following use of tyrosine kinase receptor inhibitors (including imatinib, sunitinib, lapatinib, gefitinib, and erlotinib). Relapse of injury is common upon re-exposure to the drug, but may not recur upon switching to other kinase receptor inhibitors. Probability Score: B (likely to cause clinically significant acute liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of lapatinib during lactation. Because lapatinib binds to plasma proteins at a rate exceeding 99%, its concentration in breast milk may be low. However, its half-life is approximately 24 hours, which may allow it to accumulate in the infant. Furthermore, lapatinib is used in combination with capecitabine, which may increase the risk to the infant. The manufacturer recommends discontinuing breastfeeding during lapatinib treatment and for one week after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
Highly binds to albumin and α-1 acid glycoprotein (>99%)

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Interactions
Grapefruit products should be avoided as they may lead to elevated plasma lapatinib concentrations. In patients receiving concurrent treatment with lapatinib and paclitaxel (CYP2C8 and P-gp substrates), systemic paclitaxel exposure (24-hour AUC) increased by 23%. However, the manufacturer notes that these data may underestimate the potential increase in paclitaxel exposure during combination therapy due to limitations in the study design. Concomitant use of lapatinib with oral digoxin (a P-gp substrate) can increase digoxin systemic exposure (AUC) by approximately 2.8-fold. For patients taking digoxin, serum digoxin concentrations should be measured before initiating lapatinib treatment and monitored throughout the combination therapy period. If serum digoxin concentrations exceed 1.2 ng/mL, the digoxin dose should be reduced by 50%. Because lapatinib may cause QT interval prolongation, caution should be exercised when using lapatinib in patients receiving concomitant therapy with other drugs known to prolong the QT interval (e.g., antiarrhythmic drugs). For more complete data on drug interactions of lapatinib (10 items in total), please visit the HSDB record page.

[1]. The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther. 2001 Dec;1(2):85-94.

[2]. Lapatinib, a Dual Inhibitor of Epidermal Growth Factor Receptor (EGFR) and HER-2, Enhances Radiosensitivity in Mouse Bladder Tumor Line-2 (MBT-2) Cells In Vitro and In Vivo. Med Sci Monit. 2018 Aug 20;24:5811–5819.

Lapatinib dimethylbenzenesulfonate monohydrate belongs to the quinazoline class of compounds. Lapatinib dimethylbenzenesulfonate is the dimethylbenzenesulfonate of lapatinib, a synthetic, orally effective quinazoline compound with potential antitumor activity. Lapatinib reversibly blocks the phosphorylation of epidermal growth factor receptor (EGFR), ErbB2, and Erk-1, Erk-2, and AKT kinases; it also inhibits cyclin D levels in human tumor cell lines and xenografts. EGFR and ErbB2 are associated with the growth of various tumors. It is a quinazoline derivative that inhibits epidermal growth factor receptor and HER2 (receptor, ERBB-2) tyrosine kinase. It is used to treat advanced or metastatic breast cancer with HER2 overexpression.

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Drug Indications
Tyverb is indicated for the treatment of HER2 (ErbB2) overexpressing breast cancer: in combination with capecitabine for patients with advanced or metastatic breast cancer whose disease has progressed after prior anthracycline, taxane, or trastuzumab treatment; in combination with trastuzumab for patients with hormone receptor-negative metastatic breast cancer whose disease has progressed after prior trastuzumab treatment or combination chemotherapy; and in combination with an aromatase inhibitor for postmenopausal women with hormone receptor-positive metastatic breast cancer who are currently not receiving chemotherapy. Patients in the registration study have not previously received trastuzumab or an aromatase inhibitor treatment. There are currently no data on the efficacy of this combination therapy relative to trastuzumab plus an aromatase inhibitor in this patient population.

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Therapeutic Use
Anti-tumor drug; Protein kinase inhibitor
Tyverb in combination with capecitabine is indicated for the treatment of HER2 overexpressing patients with advanced or metastatic breast cancer who have previously received anthracycline, taxane, or trastuzumab treatment. Usage Restrictions: Patients should experience disease progression during trastuzumab treatment before initiating Tykerb in combination with capecitabine. /Included in US Product Label/
Tykerb in combination with letrozole is indicated for the treatment of postmenopausal women with HER2-overexpressing hormone receptor-positive metastatic breast cancer who are receiving hormone therapy. /Included in US Product Label/
Exploring Treatments: Although effective HER2-targeting drugs are available, new combination therapy strategies need to be explored for HER2-overexpressing breast cancer patients whose tumors have developed resistance. To develop new treatment strategies, we investigated the combined effects of the oral isoform-selective histone deacetylase type I inhibitor entinostat and the HER2/EGFR dual tyrosine kinase inhibitor lapatinib in HER2-positive breast cancer cells. We evaluated the synergistic effects and mechanisms of the combination therapy using CellTiter Blue assays, flow cytometry, growth-independent assays, quantitative real-time PCR, small interfering RNA, Western blotting, and a breast fat pad xenograft mouse model. Studies have found that, compared with entinolide or lapatinib alone, the combination of the two drugs synergistically inhibited cell proliferation (P < 0.001), reduced in vitro colony formation (P < 0.05), and significantly reduced tumor volume or inhibited tumor growth in two xenograft mouse models (BT474 and SUM190) (P < 0.001). The synergistic antitumor activity of the entinolide/lapatinib combination is attributed to the downregulation of phosphorylated Akt, which activates the transcriptional activity of FOXO3, thereby inducing the expression of Bim1 (a pro-apoptotic protein containing a BH3 domain). Furthermore, entinolide enhanced the sensitivity of trastuzumab/lapatinib-resistant HER2-overexpressing cells to the trastuzumab/lapatinib combination and strengthened its antiproliferative effect, superior to either single-drug or dual-drug combination therapy. The evidence provided in this study suggests that the combination of entenostatin and the HER2-targeting drug lapatinib enhances antitumor activity and induces apoptosis through FOXO3-mediated Bim1 expression. …These results support clinical trials of entenostatin, lapatinib, and trastuzumab in combination therapy for HER2-overexpressing breast cancer patients resistant to trastuzumab.

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Drug Warning
/Black Box Warning/ Warning: Hepatotoxicity. Hepatotoxicity has been observed in both clinical trials and post-marketing experience. Hepatotoxicity can be serious, and deaths have been reported. The cause of death is unknown.
Hepatotoxicity (ALT or AST > 3 times the upper limit of normal, total bilirubin > 2 times the upper limit of normal) has been observed in both clinical trials (<1% of patients) and post-marketing experience. Hepatotoxicity can be serious, and deaths have been reported. The cause of death is unknown. Hepatotoxicity may occur within days to months after the start of treatment. Liver function (transaminases, bilirubin, and alkaline phosphatase) should be monitored before starting treatment, every 4 to 6 weeks during treatment, and as clinically necessary. If liver function changes are severe, tepatinib treatment should be discontinued and tepatinib should not be reintroduced. Lapatinib may cause harm to the fetus; animal studies have shown it can cause fetal malformations, miscarriage, and death of pups within days of birth. Pregnancy should be avoided during treatment. If lapatinib is used during pregnancy, or if a patient becomes pregnant while using the drug, the patient should be informed of the potential harm to the fetus. FDA Pregnancy Risk Classification: D / Clear evidence of risk. Human studies, research data, or post-marketing data all indicate a risk to the fetus. However, the potential benefits of using this drug may outweigh the potential risks. For example, this drug may be acceptable in life-threatening situations or when a patient has a serious illness and other safer medications are unavailable or ineffective. / For more complete data on lapatinib (14 total), please visit the HSDB record page.

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Pharmacodynamics
Lapatinib is a small molecule belonging to the 4-phenylaminoquinazoline kinase inhibitor class. Lapatinib is an anticancer drug developed by GlaxoSmithKline (GSK) for the treatment of solid tumors such as breast cancer and lung cancer. On March 13, 2007, the U.S. Food and Drug Administration (FDA) approved GW2016 in combination with the chemotherapy drug capecitabine for the treatment of patients with advanced metastatic breast cancer. Epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinase are targets of multiple mechanisms in current cancer treatment. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains, with IC50 values of 10.2 nM and 9.8 nM for purified EGFR and ErbB-2, respectively.
This report describes the efficacy of GW2016 in cell growth assays of human tumor cell lines overexpressing EGFR or ErbB-2, including HN5 (head and neck cancer), A-431 (vulvar cancer), BT474 (breast cancer), CaLu-3 (lung cancer), and N87 (gastric cancer). Normal human foreskin fibroblasts, non-neoplastic epithelial cells (HB4a), and non-overexpressing tumor cells (MCF-7 and T47D) served as negative controls. Three days after compound exposure, the mean IC50 values for growth inhibition in EGFR and ErbB-2 overexpressing tumor cell lines were < 0.16 μM. The mean selectivity of the compound for tumor cells relative to human foreskin fibroblast cell lines was 100-fold. Western blot analysis validated the inhibition of EGFR and ErbB-2 receptor autophosphorylation and the phosphorylation of its downstream regulator AKT in the BT474 and HN5 cell lines. To evaluate cytotoxicity and growth inhibition, growth assays were performed on HN5 and BT474 cells after transient exposure to GW2016. Cells were treated with five different concentrations of GW2016 for 3 days, and cell growth was monitored for 12 days after compound removal. GW2016 reached a concentration that inhibited excessive cell growth in each tumor cell line. Furthermore, growth arrest and cell death were observed in parallel experiments and detected by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 inhibited tumor xenograft growth in HN5 and BT474 cells in a dose-dependent manner at oral doses of 30 and 100 mg/kg twice daily, with the 100 mg/kg dose completely inhibiting tumor growth. In summary, these results indicate that GW2016 possesses excellent inhibitory activity against tumor cells and exhibits selectivity, suggesting that GW2016 may have therapeutic value for patients with tumors overexpressing EGFR or ErbB-2. [1]

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Background: This study aimed to evaluate the effect of lapatinib (a dual inhibitor of epidermal growth factor receptor (EGFR) and HER-2) on the radiosensitivity of mouse bladder tumor cell line 2 (MBT-2) (in vitro and in vivo). Materials/Methods: MBT-2 cells were pretreated with 200–1000 nM lapatinib for 30 min and then irradiated with 2.5–10 Gy for 30 min. Cell viability was assessed using a colony formation assay. The expression of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), phosphorylated HER-2 (p-HER2), and the apoptosis marker PARP was detected by Western blot. MBT-2 cells were subcutaneously injected into a C3H/HeN mouse tumor xenograft model; mice were divided into four groups and received lapatinib (200 mg/kg), irradiation (15 Gy), a combination of both, and a vector (control group), respectively. Results showed that lapatinib pretreatment combined with radiotherapy reduced MBT-2 cell survival and inhibited radiation-activated p-EGFR and p-HER-2 levels. The combined index (CI) of MBT-2 cells treated with 10 Gy radiation and 1000 nM lapatinib was <1, indicating a synergistic effect. Increased γ-H2AX expression suggested enhanced apoptosis. In a mouse model of tumor xenograft, administration of lapatinib (200 mg/kg/day) daily for seven consecutive days, combined with radiotherapy on day four, significantly inhibited tumor growth compared to radiotherapy alone. Conclusion: Lapatinib treatment enhances radiosensitivity in in vitro and in vivo mouse bladder cancer models by reducing radiation-mediated EGFR and HER-2 activation and inducing apoptosis through DNA damage.

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In the mid-1980s, it was recognized that overexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) could adversely affect the prognosis of some cancer patients. Because EGFR and HER2 are key regulators of cell growth, differentiation, and survival, it is believed that inhibiting these receptors can block downstream signaling, thereby exerting an anti-proliferative effect. Indeed, by the late 1980s, researchers had developed some of the earliest targeted inhibitors of tyrosine kinase activity. It is against this backdrop that we began working on developing potential disease therapies using a small molecule strategy to selectively target EGFR and HER2. The challenge in developing small molecule EGFR/HER2 inhibitors lies in determining their potency and selectivity against the kinase domains. Several key components of our research have driven this drug discovery effort. First, novel chemical methods have prepared a large number of compounds for testing. Second, a comprehensive kinase biochemical screening platform has enabled us to examine the effects of compounds on multiple kinase targets. Third, we have constructed a cell-based screening platform that includes cell lines dependent on the EGFR or HER2 signaling pathway, as well as suitable control cell lines. This cell screening platform not only automates the assessment of the potency and selectivity of molecules in the complex cellular environment but also allows us to study downstream regulators of EGFR and HER2, thus linking EGFR and HER2 inhibition to cell cycle arrest and apoptosis. The final component, the in vivo xenograft model, enabled us to continue this research using pharmacokinetic and pharmacodynamic biomarkers. Our collaboration with a highly dedicated and proactive team of scientists ultimately led to the groundbreaking discovery of GW2016 (also known as GW572016), a compound that later evolved into the cancer treatment lapatinib. Lapatinib's selectivity for the HER2 and EGFR kinase domains, and its activity in HER2-overexpressing cell lines (e.g., breast and gastric cancer) and EGFR-overexpressing cell lines (e.g., head and neck cancer), laid the foundation for testing lapatinib in specific patient populations. In 2007, the U.S. Food and Drug Administration approved lapatinib in combination with capecitabine for the treatment of advanced or metastatic HER2-overexpressing breast cancer, providing a new treatment option for patients whose disease has progressed after treatment with trastuzumab (a humanized monoclonal antibody targeting the extracellular domain of HER2). Ongoing clinical trials are investigating the activity of lapatinib in HER2-overexpressing breast cancer, HER2-overexpressing gastric cancer, and head and neck cancer. Looking ahead, treatment strategies targeting HER2 and EGFR will be similar to those used in most future cancer therapies (i.e., in combination with other drugs). Recent clinical evidence suggests a synergistic effect of dual blockade of lapatinib in combination with trastuzumab in patients with HER2-positive metastatic breast cancer. Furthermore, dual blockade of the HER2 signaling pathway is being investigated in neoadjuvant and adjuvant therapy. Other clinical studies are evaluating the efficacy of HER2 and EGFR-targeted drugs in combination with other signaling pathway agents and chemotherapy drugs. We are encouraged by the results of our efforts since we first published our findings on lapatinib 10 years ago and will continue to dedicate ourselves to improving the lives of cancer patients. https://aacrjournals.org/mct/article/10/11/2019/90946/The-Discovery-of-Lapatinib-GW572016-Commentary-on

C43H44CLFN4O11S3

943.48

942.184

C, 55.81; H, 4.57; Cl, 3.83; F, 2.05; N, 6.05; O, 17.29; S, 10.39

388082-78-8

Lapatinib;231277-92-2;Lapatinib ditosylate;388082-77-7; Lapatinib ditosylate monohydrate;388082-78-8;Lapatinib-d4;1184263-99-7;Lapatinib tosylate;1187538-35-7;Lapatinib-d7 dihydrochloride;Lapatinib-d5;2748212-14-6;Lapatinib-d4-1;1184264-15-0

11557040

Light yellow to yellow solid powder

1.403 g/mL (20.84 °C)

750.7ºC at 760 mmHg

237 - 239 °C

68 °F

n20/D 1.5520(lit.)

12.264

5

16

13

63

1100

0

XNRVGTHNYCNCFF-UHFFFAOYSA-N

InChI=1S/C29H26ClFN4O4S.2C7H8O3S.H2O/c1-40(36,37)12-11-32-16-23-7-10-27(39-23)20-5-8-26-24(14-20)29(34-18-33-26)35-22-6-9-28(25(30)15-22)38-17-19-3-2-4-21(31)13-19;2*1-6-2-4-7(5-3-6)11(8,9)10;/h2-10,13-15,18,32H,11-12,16-17H2,1H3,(H,33,34,35);2*2-5H,1H3,(H,8,9,10);1H2

N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]furan-2-yl]quinazolin-4-amine;4-methylbenzenesulfonic acid;hydrate

Lapatinib Ditosylate; 388082-77-7; Tykerb; Lapatinib (ditosylate); Lapatinib (GW-572016) Ditosylate; N-(3-CHLORO-4-((3-FLUOROBENZYL)OXY)PHENYL)-6-(5-(((2-(METHYLSULFONYL)ETHYL)AMINO)METHYL)FURAN-2-YL)QUINAZOLIN-4-AMINE BIS(4-METHYLBENZENESULFONATE); Lapatinib ditosylate anhydrous; 4WK72K94MC;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : 50 mg/mL (53.00 mM)

Solubility in Formulation 1: 2.5 mg/mL (2.65 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)