LAPATINIB NANO-DELIVERY SYSTEMS: A PROMISING FUTURE FOR BREAST CANCER TREATMENT
Gunjan Vasant Bonde, Sarita Kumari Yadav, Sheetal Chauhan, Pooja Mittal, Gufran Ajmal, Sathish Thokala & Brahmeshwar Mishra
To cite this article: Gunjan Vasant Bonde, Sarita Kumari Yadav, Sheetal Chauhan, Pooja Mittal, Gufran Ajmal, Sathish Thokala & Brahmeshwar Mishra (2018): LAPATINIB NANO-DELIVERY SYSTEMS: A PROMISING FUTURE FOR BREAST CANCER TREATMENT, Expert Opinion on Drug Delivery, DOI: 10.1080/17425247.2018.1449832
To link to this article: https://doi.org/10.1080/17425247.2018.1449832
Accepted author version posted online: 09 Mar 2018.
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Publisher: Taylor & Francis
Journal: Expert Opinion on Drug Delivery
DOI: 10.1080/17425247.2018.1449832
LAPATINIB NANO-DELIVERY SYSTEMS: A PROMISING FUTURE FOR BREAST CANCER TREATMENT
Gunjan Vasant Bonde1, Sarita Kumari Yadav1,2, Sheetal Chauhan3, Pooja Mittal1, Gufran Ajmal1, Sathish Thokala1, Brahmeshwar Mishra*1
⦁ Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology, Banaras Hindu University, Varanasi, India
⦁ Moti Lal Nehru Medical College, Allahabad, India
⦁ Department of Pharmacology, Melaka Manipal Medical College, Manipal University, Karnataka, India
Corresponding Author’s name: Brahmeshwar Mishra
Address: Professor of Pharmaceutics, Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology, Banaras Hindu University, Varanasi, Pin-221005 (UP), India. Tel: +91-542-6702748; Fax: +91-542- 2368428
E-mails of all authors: [email protected], [email protected], [email protected] ,
[email protected] , [email protected] , [email protected] ,
Funding:
This paper was not funded.
Declaration of interest:
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Peer reviewers on this manuscript have no relevant financial or other relationships to disclose
Author contributions:
GV Bonde collected all the literature for this review and both edited and prepared the manuscript for submission. B Mishra provided guidance for the collection of literature information and manuscript editing. SK Yadav provided guidance for arrangement of collected literature information. S Chauhan and P Mittal assisted in language and paragraph editing, G Ajmal assisted in the overall editing of the manuscript as per journal guidelines. S Thokala assisted in editing references as per journal guidelines.
ABSTRACT
Introduction: Breast cancer stands the second prominent cause of death among women. For its efficient treatment, Lapatinib (LAPA) was developed as a selective tyrosine kinase inhibitor of receptors, overexpressed by breast cancer cells. Various explored delivery strategies for LAPA indicated its controlled release with enhanced aqueous solubility, improved bioavailability, decreased plasma protein binding, reduced dose and toxicity to the other organs with maximized clinical efficacy, compared to its marketed tablet formulation.
Areas Covered: This comprehensive review deals with the survey, performed through different electronic databases, regarding various challenges and their solutions attained by fabricating delivery systems like nanoparticles, micelle, nanocapsules, nanochannels, and liposomes. It also covers the synthesis of novel LAPA-conjugates for diagnostic purpose.
Expert Opinion: Unfortunately, clinical use of LAPA is restricted because of its extensive albumin binding capacity, poor oral bioavailability, and poor aqueous solubility. LAPA is marketed as the oral tablet only. Therefore, it becomes imperative to formulate alternate efficient multiparticulate or nano-delivery systems for administration through non-oral routes, for active/passive targeting, and to scale-up by pharmaceutical scientists followed by their clinical trials by clinical experts. LAPA combinations with capecitabine and letrozole should also be tried for breast cancer treatment.
Keywords: Active Targeting, Capecitabine, Letrozole, Nano-Delivery System, Passive Targeting, Tyrosine Kinase.
⦁ Introduction
Worldwide, cancer has become the second leading cause of death. There were 14.1 million new cases in 2012, which is speculated to increase to 17.1 million cases in 2020 [1]. The most prevalent cancers are lung, liver, colorectal, stomach and breast cancer. The breast cancer (BC) took fifth place in all cancers and second in women cancer, being responsible for 571,000 deaths out of 8.8 million cancer deaths in 2015 [2]. The occurrence rate of BC ranged from 27 per 100,000 in Eastern Asia and Middle Africa to 92 in Northern America. It has high incidence, approximately 1.67 million cases reported in 2012 which contributes 25% of all types of cancers. The prediction of BC prevalence is about 1.98 million cases in 2020 [3].
⦁ Classification of BC and its treatment
BC is one of the heterogeneous diseases [4] and therefore, the correct assessment of prognosis and appropriate treatment was achieved by categorizing it by the criteria like patient age, tumor size, status of axillary lymph node, histological features, a status of hormone receptor, and human epidermal growth factor receptor (HER2) status [5]. Considering histology of tumors, BC is broadly classified as in situ and invasive carcinoma. Detailed classification is shown in Figure 1 [6,7]. Further, in light of breast-conserving surgical procedures, BC was classified by considering status molecular markers such as HER2, progesterone receptor (PR), p53 and estrogen receptor (ER), etc. [8,9] which help not only for the selection of the appropriate therapy but also to predict whether the patient is likely to respond to the selected targeted cancer therapy [10]. Beyond traditional hormone receptor, positive/negative types, other molecular techniques like microarray-based gene expression analysis and unbiased hierarchical clustering techniques [11] were exploited to identify and classify several newer intrinsic molecular subtypes for proper molecular classification of BC, summarized in Figure 2(a) [10,12]. Besides molecular classification, an emerging area of research in the field of BC is cancer stem cells (CSC) which further, come up with the functional classification system of breast cancer, depicted in Figure 2(b,c) [10,13,14].
Nowadays, various treatments are available for the BC management. The conventional therapies are classified as local therapy, consisting of either surgery or radiation therapy,
whereas systemic therapies involve chemotherapy, gene and hormonal therapy [15,16]. The classification of practiced therapies is summarized in Figure 3[17,18]. For effective management of BC, these therapies are used as either single or combination of two or more, depending upon the stage of cancer progression in a patient [19]. With the development in the area of novel drugs and their delivery systems, the shift from “same for all” treatment to the era of “targeted or personalized” treatments has been observed [20,21].Targeted therapies exhibit the principles of either drug targeting, active targeting or passive targeting as illustrated in Figure 4. Drug targeting is achieved by designing the drug complementary to the receptor overexpressed predominantly by cancer cells, e.g. Epidermal Growth Factor Receptor (EGFR) and HER2 [22-24]. Such drugs include tyrosine kinase inhibitors (TKIs) like Lapatinib (LAPA), Sunitinib, Erlotinib, monoclonal antibodies like Trastuzumab, etc. [25].
⦁ Lapatinib related information
On March 13, 2007, United States Food and Drug Administration approved LAPA, in combination with capecitabine (Xeloda®), for the treatment of HER2 overexpressing advanced and metastatic BC, underwent prior treatment with anthracycline, taxane, and trastuzumab [26,27] . Whereas, on January 29, 2010, LAPA was approved as a combination with letrozole for the treatment of BC patients preferably postmenopausal women with hormone receptor positive metastatic BC overexpressing the HER2 receptor and for whom hormonal therapy is suggested [26]. Tykerb® is the unique tablet dosage form of LAPA marketed by GlaxoSmithKline (pharmaceutical company) with a dose of 250 mg.
⦁ Pharmacokinetics
LAPA is a Biopharmaceutical Classification Class II drug [28] having poor aqueous solubility (7µg/ml) [29]. The restricted dissolution in gastrointestinal fluidsresults in poor and erratic absorption from the gastrointestinal tract leading to its low oral bioavailability. However, the bioavailability can be enhanced by 325% after co-administration with a high fat meal as compared with fasting conditions [30]. Unfortunately, LAPA has about 99% binding tendency to albumin and alpha-1 glycoprotein in the blood, which further reduces the availability of free drug at the tumor site [31]. Therefore, to achieve effective drug concentration at the intended site, it must be administered as a large daily dosethat limits the clinical use of LAPA. In Phase I trials, the steady state pharmacokinetic parameters like AUC, tmax , Cmax and Cmin at 20th day were found to be 14.3 h.g/mL, 4 h, 1.22 and 0.30 g/mL,
respectively, post-oral administration of 1200 mg dose in heavily pretreated patients with metastatic carcinoma [32]. The drug is eliminated significantly by hepatic metabolism, however around 27% and <2% of oral dose is eliminated via feces and urine, respectively [33]. The complete drug profile is given in Table 1.
⦁ Pharmacodynamics
LAPA is a member of the 4-anilinoquinazoline family of TKIs. It targets and binds potently and reversibly to the intracellular TK domains of both receptors, HER2 and EGFR. This inhibition interrupts downstream MAPK and PI3K signaling pathways and proliferation both in vitro and in vivo [34]. Since few recent studies reported the overexpression of EGFR in triple negative breast cancer (TNBC), it has gained particular attention in the management of TNBC, characterized by all ER/PR/HER2 negative [35]. The larger dose leads to adverse effects like severe diarrhea, nausea, and rash [36]. The combination of LAPA with paclitaxel and capecitabine increased the risk of hepatotoxicity and were reported to increase aspartate transaminase and total bilirubin by >3 times and >2 times the upper limit of normal, respectively [37,38].
Multidrug resistance (MDR) is well known fact responsible for lowering the therapeutic efficacies of certain drugs because of the resistance induced due to the eflux of such drugs from inside to outside of the cell by certain transporters like ABC transporters and P-glycoprotein (P-gp) in case of PTX [39], BCRP and P-glycoprotein (P-gp) in case of DOX [40]. Similarly, various factors like the mutation in the BCR-ABL kinase domains, overexpression of the multidrug-resistant P-glycoprotein (MDR-1) and amplification of BCR-ABL1 [41,42] are responsible for the resistance to Imatinib, which selectively inactivate
the BCR-ABL tyrosine kinase in cancer cells [43,44]. LAPA also has been reported to have an inhibitory action on various MDR transporters via interaction with the substrate-binding site [39,45,46]. Therefore, the combination of such drugs with LAPA had been explored and proven to be effective in the treatment of MDR cancer [40,47-52]. Similarly, the combination of LAPA and Imatinib was also reported as a better treatment for TNBC [53].
All these facts necessitate the revolution of the drug delivery systems (DDS) by;
⦁ Targeting the tumors precisely by either active or passive targeting,
⦁ Enhancing the oral bioavailability,
⦁ Developing the injectable DDS for systemic delivery, local delivery etc.
⦁ Encapsulation of drug to alleviate its adsorption to plasma proteins.
In the present review, we attempted to give elaborate information to shed light on the recent developments in the targeted LAPA delivery systems (Table 2) for addressing the above-said inherent problems and effective management of the BC.
⦁ Formulation approaches for LAPA
Developments in nanotechnology come up with various types of nanocarriers with some excellent characteristics which are anticipated to solve the problems like poor aqueous solubility, lower bioavailability, and poor targeting ability, etc., faced by conventional DDS. The characteristics of nanocarriers and their particular use for designing DDS are listed in Table 3 [29,49,51,54-60]. The nanocarriers include nanoparticles (NPs), nanocapsules, polymeric micelle (PM), etc. The nanocarriers surface can be amended for active targeting to tumors by exploiting the principle of ligand-receptor interaction between ligand harbored to the surface of DDS and specific receptors on cancer cell surface [58]. However, passive targeting to tumors could be accomplished by taking advantage of their nano size, enabling them for easy crossing the leaky vasculature of tumors, commonly referred as Enhanced Permeation and Retention (EPR) effect [54,55]. Principles of targeting are illustrated in Figure 5. Considering these benefits of nanocarriers, several research groups attempted to formulate various types of nanocarrier based DDS for better management of BC and their possible schematic diagrams are depicted in Figure 4
⦁ Nanoparticles
LAPA was reported to have a high albumin binding affinity [31]. By exploiting this inherent property, Wan et al. [61,62] prepared Human Serum Albumin (HSA) based nanoparticles, employing NAB technology [63], for intravenous administration. The prepared freeze-dried LAPA nanoparticles (LNs) had mean particle size 140 nm with narrow particle size
distribution and good encapsulation efficiency (EE) (nearly 85%). They showed sustained release of drug during five day incubation period, with approximately 60% release in diluted mouse plasma whereas it was less than 50% in phosphate buffer saline (PBS). Importantly, better inhibition of growth of carcinoma spheroids by LNs, in comparison with free drug, indicated their higher penetrating power.
In vivo pharmacokinetic studies in rats revealed that incorporation of LAPA in nanoparticles significantly prolonged elimination half-life with higher AUC0-48h, and extended mean residence time as compared to that of lapatinib solution (LS). The absolute bioavailability was found to be 8.08 fold higher than that of the Tykerb®. These experimental findings suggested their longer systemic circulation. As LAPA is an inhibitor of both HER2 and EGFR, the research group investigated the activity of LNs against two cell lines namely 4T1 (EGFR overexpressing) TNBC cell line and SKBr3 (HER2 overexpressing) cell lines.
The cellular uptake of LNs was confirmed as energy dependent and temperature sensitive. Cell apoptosis study of nanoparticles in 4T1 cell line showed higher early apoptosis rate, but overall findings suggested no significant difference in a total number of dead and apoptotic cells between LS and LNs groups. On the other hand, in case of SKBr3 cell line, the treatment with LNs significantly increased the apoptosis as compared to LS. Hence, suggesting a higher cytotoxic effect of LNs, that was in agreement with the findings of MTT assays.
Further, the LNs were investigated for in vivo tumor growth inhibition studies in both the cell lines. The results revealed excellent antitumor efficacy of LNs (3 mg/kg i.v.), compared to that of treatment with Tykerb® (100 mg/kg p.o.) and LS (10 mg/kg i.v.). Inhibition of micrometastasis to lungs was observed in 4T1 cells but extravasation of tumor cells found around vessels at low doses of LNs. However, no micrometastasis and extravasation were observed at a higher dose of LNs (total dose was 10% of Tykerb® group) suggesting the dose dependent activity of the drug. All these results demonstrated the potential of LAPA-loaded HSA nanoparticles as a promising DDS for treatment of TNBC as well as HER2 positive breast cancer [61,62].
Another attempt for effective delivery of LAPA was carried out by Huo et al. [56]. They investigated polymeric lipid hybrid nanoparticles (PLPT) as DDS. The nanoformulation consisted of polymeric core made up of (poly[lactide-co-glycolide]-D-a-tocopheryl
polyethylene glycol 1000 succinate [PLGA–TPGS]). Itwas previously synthesized using ring opening polymerization method, and then enveloped by a PEGylated lipid layer of 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000 (DSPE-PEG) to strengthen the structural integrity. The prepared PLPT had a spherical shape with an average diameter of 160±3.2 nm and narrow size distribution while loading capacity and entrapment efficiency was 15% and >80%, respectively. In vitro drug release studies demonstrated a sustained release of a drug upto 120 hs which could be the reason for the reduction in dose and dose frequency, thereby improves the therapeutic efficacy of a drug. PLPT also exhibited prolonged half-life and five-fold higher tumor accumulation than free drug, which was referred to hydrophilic PEG shell and reduced uptake by the reticuloendothelial system (RES) [64].
Further, PLPT were subjected to the in vitro cellular uptake and cell apoptosis analysis, by employing MCF-7 cancer cell lines as a model. From the results, a remarkable time- dependent uptake of PLPT was observed by cells during incubation which may enhance the cell cytotoxicity. Higher cell apoptosis in the PLPT group was attributed not only to the sustained release of the drug but also to their remarkable cellular uptake by MCF-7 cells. Further, the experimental findings of antitumor efficacy studies, in MCF-7 bearing xenograft tumor models, exhibited that PLPT was a potent formulation for antitumor effect as it could reduce the cancerous tumor burden as compared to free drug significantly [56].
Some drugs like LAPA are highly bound to albumin, and hence high doses are required to elucidate the intended response. However, this problem was thought to be surmounted by encapsulation of drug in nanocarrier by shielding it from getting adsorbed on plasma proteins which may increase the free drug concentration at action site and help in reduction of effective drug dose size. Various polymers are used to encapsulate drug for the production of nanocarrier systems [65]. The carbohydrate polymer proved to have greater potentials to formulate as drug carriers owing to their nontoxic and biodegradable nature. Among carbohydrates, Mobasseri et al. [66] used dextran sulfate and chitosan to prepare nanoparticles for encapsulation of LAPA. The LNs were prepared by cross-linking the polymers by adding cross-linkers like calcium chloride, zinc sulfate, and magnesium chloride. Among these, calcium chloride showed effective size reduction of LNs than others. The prepared LNs had negative zeta potential indicating their stability since the positive zeta potential was previously reported to induce their uptake by RES [57].
Further, the LNs showed a pH-dependent biphasic pattern of drug release. The first phase of higher drug release for 4 hs was attributed to dissolution of surface adsorbed and surface proximity drug followed by the second phase of sustained release from the core of nanoparticles till 96 hs . The satisfactory cytotoxicity against BT474 was induced after administration of LNs, but it was worse as compared to LS within first 24 hs. However, the results were found to be reversed at the 48th h with higher cytotoxicity of LNs as compared to LS evidenced by low IC50 value. Thus, the dextran-chitosan nanoparticles could serve not only as better nanocarrier system but also as sustained release system for hydrophobic drugs [66].
Zhang et al. [67] fabricated LAPA incorporated lipoprotein like nanoparticles (LTNP) with a core consisting LAPA bound albumin whereas lipid corona formed by Egg Yolk Lecithin (EYL). LTNP were prepared by dissolving the drug in the mixture of ethanol and deionized water which was then added to the solution of EYL in dichloromethane. This prepared mixture was then added dropwise to the solution of bovine serum albumin in deionized water. Further, the organic solvents were removed by rotary evaporator to get LTNP.
Prepared LTNPs were found to have a mean particle size of 62.1 nm with narrow polydispersity index (PDI) of 0.21 with a spherical shape. Cellular uptake of LTNPs was investigated in BT-474 cell line. Experimental findings denoted that cellular uptake was mediated by clathrin-mediated endocytosis and macropinocytosis. Tumor distribution of LTNPs was performed on U87 glioma tumor-bearing mice. The results revealed the effective distribution of LTNPs throughout the tumor which was attributed to the leaky vasculature in the tumor and EPR effect [54]. The LTNPs (20 μg/mL) brought about a marked cell arrest at a G0/G1 phase in BT-474 cells than LAPA suspension [67].
⦁ Core-shell Nanoparticles (CSNs)
Entrapment of LAPA in the core of CSNs shields its interaction with aqueous environment and may enhance its water solubility, thereby, may improve its bioavailability. On this basis, Gao et al. [29,60] formulated the CSNs having a hydrophobic core where hydrophobic drugs can be entrapped while hydrophilic shell can solubilize them in an aqueous environment. LAPA incorporated core-shell nanoparticles (LTNP) were prepared by employing NAB technology [63]. The prepared LTNPs found to have a spherical shape with mean particle
size of 66.8nm, and their core-shell structure was confirmed by cryo-TEM. LTNPs exhibited enhanced aqueous solubility of LAPA from 0.007 mg/mL to 10 mg/mL.
Further, cellular uptake and identification of uptake mechanism of LTNPs were identified as energy dependent endocytosis viz. clathrin-mediated endocytosis, caveole-mediated endocytosis, and macropinocytosis. In U87 cells, uptake of LTNPs induced the inhibition of proliferation by G2 phase arrest. Glioma distributions of LTNPs were studied by using in vivo imaging technique. The results showed their higher accumulation in the glioma as compared to normal brain tissue due to EPR effect. These results were in agreement with the pharmacokinetic studies, showing the glioma/brain concentration ratio of LAPA was approx.
30.3 in LTNP group which was much higher than that of Tykerb® group and less than 1.5, at the end of 8 hs.
The pharmacodynamic study, glioma-bearing mice, revealed that LTNPs could effectively increase the median survival time at a cumulative dose of 40 mg/kg. It is noteworthy to mention that the administered LTNPs dose was only 5% that of Tykerb®. Hence, LTNPs could serve as an effective delivery vehicle to target glioma with significant dose reduction, alleviating the possibility of side effects [29,60].
⦁ Nanocapsules
LAPA also exhibited to have an inhibitory effect on the function of ABC transporters, including P-gp [39]. Hence, Vergara et al. [48] had hypothesized to combine paclitaxel (PTX) with LAPA to overcome MDR for PTX. The sonication-assisted layer-by-layer (SLBL) technique was employed to incorporate both drugs in different layers constituting a nanocapsule. For the formation of LBLs, chitosan (polycation) and alginic acid (polyanion) were chosen and were prepared by washless LBL method [68]. Briefly, PTX, chitosan, and ammonium bicarbonate were mixed with distilled water and then ultrasonicated in a water/ice bath to prepare PTX/chitosan nanocores. After 45 min sonication, alginic acid solution (1 mg/mL) was added and sonicated to form PTX/chitosan/alginic acid core-shell structure. Chitosan and alginic acid solutions were sequentially added to make a three layered capsule wall of PTX/(chitosan/alginic acid)3 composition (LBL-PTX). To above first capsule, LS was added followed by alginic acid solution to form capsule shell containing LAPA whose diameter was found to be around 250 nm. Prepared nanocapsules were investigated for anti- tumor activity by MTT assay using OVCAR-3, multidrug-resistant ovarian cancer cell line.
Results depicted significant growth inhibition of cells after treatment with LBL-PTX (1.5 ng/mL) as compared to that of free PTX. Moreover, combination therapy of LBL-PTX and LAPA demonstrated the enhanced cytotoxic efficacy of nanocapsules when compared with that of free PTX and LBL-PTX. At the same time, net negative charge on nanocapsules could prove advantageous for tumor penetration and accumulation indicating their higher potential for anticancer treatment [48,69].
⦁ Polymeric Micelles
Wang et al. [40] fabricated PMs consisting of poorly-water soluble drugs like doxorubicin (DOX) and LAPA combination to treat MDR-BC. They synthesized the novel polymer Poly(ethylene glycol)-block-poly (2-methyl-2-benzoxycarbonylpropylene carbonate) (PEG- PBC) which was used for the formulation of combination micelle by film dispersion method. The low critical micelle concentration (CMC) of synthesized polymer (1.5 mg/L) suggested its high dynamic stability. The micelles were spherical with mean diameter of 100 nm. Further, micelles were investigated for in vivo anticancer efficacy studies using drug resistant MCF-7/ADR cells. The results corroborated that DOX free, as well as DOX micelle, showed tumor growth inhibition but their combination with LAPA showed potent anticancer activity. Hence, it could conclude that LAPA significantly increased anticancer activity as well as intracellular accumulation of DOX which was referred to inhibition of BCRP and P-gp by LAPA, which was in agreement with literature [40].
About 30% of malignant tumors of breast carcinoma patients are HER2 positive [70,71]. However, higher resistance against PTX was attributed to overexpression of HER2 [72]. LAPA was reported not only to inhibit the progression of HER2 overexpressing BC tumors
[73] but also effectively enhance the sensitivity of tumor cells towards PTX, as demonstrated in phase II and III clinical trials [39,74,75]. With this background, Wei et al. [49] hypothesized the micellar co-delivery of LAPA and PTX to enhance anticancer efficacy against HER2-positive BC through their synergistic activity. The polymer needed for micelle preparation was synthesized by conjugating LAPA with PEG and poly(lactic acid) to enhance incorporation of LAPA in micelle (PM-LP). Micelles were prepared by thin film hydration method and characterized for particle size, loading capacity, zeta potential, encapsulation efficiency. The results revealed that PM-LP had average particle size of 25.80±0.47 nm with zeta potential -3.17±0.15 mV demonstrating that presence of LAPA partially neutralize the negative charge of a micellar surface which was more negative in case of PM-PTX. PM-LP
could be an excellent delivery vehicle as they were reported to have high drug loading capacity of 14.82±2.16% as well as encapsulation efficiency of 84.21±9.83%. Experimental findings of cytotoxicity test, employing SKBr-3 (HER2 positive) cancer cell line, exhibited the enhancement of anticancer activity of PTX by the synergistic effect of LPT, as evidenced by the reduction in IC50 (Inhibitory Concentration) value of PM-LP to less than half as compared to that of PM-PTX. Results of apoptosis assay were also consistent with findings of in vitro cytotoxicity as enhanced apoptosis was observed in cells treated with combined micelle PM-LP. All these facts suggested that the PM-LP could be the better DDS to treat HER2 positive breast cancer [49].
Similarly, Li et al. [47] synthesized the di-block polymer of PEG with poly(2-methyl-2- carboxyl-propylene carbonate-graft-dodecanol) (PEG-PCD) and was further used for the preparation of PM for loading of LAPA and PTX for the treatment of MDR prostate cancer. Prepared micelles were found to have approx. diameter 60 nm with almost 100% drug loading. The reversal of MDR for PTX was evidenced by results of cell cytotoxicity assays which indicated significantly reduced cell viability (35%) in the group treated with combination micelle (1µM LAPA + 0.25 µM PTX) and was further reduced to 23% during treatment with combination micelle (2µM LAPA + 0.5 µM PTX) as compared to control group. These results, for reversal of MDR for PTX, were further supported by a reduction in the IC50 value of combination well below than that of PTX alone in DU145-TXR cancer cell line. Thus, all the results indicate sensitizing behavior of LAPA on DU145-TXR to PTX. Similar results were obtained in Calcein AM assay where intracellular fluorescence intensity was reported to enhance upto 60 fold in the presence of LAPA when compared to that of in the absence of LAPA, indicating its P-gp inhibition activity and thus supported the reversal of MDR for PTX. Furthermore, the increased cell population in sub-G1 phase inferred that the combination therapy could enhance the cell apoptosis rate. Similar to in vitro studies, angiogenic activity and tumor growth significantly inhibited after treatment with combination (5mg/kg PTX +5mg/kg LAPA), as compared to monotherapy with PTX with 10 mg/kg dose. Thus, the effective dose of these two drugs could be achieved through their combination therapy for management of BC with minimal expected side effects [47].
For the preparation of micelle, pluronic especially Pluronic F127 (PF127) was well explored amphiphilic triblock polymer consisting of hydrophobic polypropylene oxide and hydrophilic polyethylene oxide chains. As discussed above, Dehghan et al. [50] attempted to deliver both, PTX and LAPA, through the PF127 micelle. The micelle was optimized to have a diameter of
approx. 64.81 nm with percent entrapment of 68.3% and 70.1% of PTX and LAPA, respectively. The smooth surface and spherical shape convey the property to escape from RES prolonging their circulation in the body and could be beneficial for passive targeting. PM exhibited the biphasic in vitro release pattern for both the drugs. The slower release of LAPA was attributed to its more hydrophobicity than PTX, in turn, more hydrophobic interactions with hydrophobic core of micelle restricting its release. The results of in vitro cytotoxicity assays revealed that the IC50 value of combination micelle for T-47D metastatic BC cell line got decreased well below that of bare PTX-LAPA solution, which were in good agreement with the literature. The mechanism for this superior cytotoxic activity of combination micelle was deduced to be due to: first- the sensitizing action of LAPA on reflux transporters increasing intracellular PTX concentration and second- direct uptake of micellar systems via endocytosis avoiding contact of bare drugs to transporters in cellular domain. On the other hand, PF 127 also supports the cytotoxicity of formulation owing to its known ATP depletion activity in cancer cells which inhibit P-gp efflux activity [76,77]. Thus, the formulated PF127 combination micelle could serve as better alternative nanocarrier system to treat metastatic breast cancer [50].
⦁ In situ Gel
One of the limitations of LAPA is its higher dose, due to poor bioavailability after oral administration and extreme binding tendency to plasma proteins notably albumin, leading to a meager fraction of administered dose to reach the site of action. For resolving these issues, Hu et al. [51] prepared a thermoresponsive in situ hydrogel, using PF 127 as a polymer, in which the nanoparticles of PTX (PTX NPs) and microparticles of LAPA (LAPA MPs) were incorporated, for local treatment of BC. The cold method [78] was employed to prepare only PTX gel (P-gel), only LAPA gel (L-gel) and the combination of both PTX and LAPA gel (PL-gel).
The morphological evaluation, using TEM and SEM, showed that PTX NPs were short rods (150-200 nm) and LAPA MPs were long rods (20 μm). The gelling temperature was between 20 to 22 ºC. The dialysis method was used to perform in vitro release studies and the results revealed that the drug release from LAPA MPs was much slower than that of PTX NPs which ascertained the higher PTX concentration at starting phase. Further, in vivo retention studies demonstrated that the diffusion of LAPA MPs was much slower than PTX NPs, after peritumoral injection of gels in BT474 tumor bearing mice models. Collectively, it could be
concluded that slow diffusion and release of LAPA MPs showed their strong retention and sustained action behavior while the rapid diffusion and release of PTX NPs showed its high concentration for a short period, peritumorally. At the same time, tumor residue analysis studies demonstrated larger accumulation of LAPA at the tumor site than that of after oral administration signifying its improved therapeutic potential.
Moreover, the calculated IC50 and CI50 (Combination Index at 50% cell growth inhibition) values, in cytotoxicity assays, confirmed the synergistic effect of the combination against BT474 cell lines, characterized by high HER2 and low P-gp expression. A synergistic effect was even more intense in MCF7/ADR cell lines, which expresse P-gp at high level in addition to HER2, confirming P-gp inhibitory action of LAPA. Hence, the findings confirmed the MDR reversal effect of LAPA. The outcome of pharmacodynamic studies proved the significant efficacy of the localized combination therapy of PTX and LAPA, having longer survival time observed by PL-gel than that in P-gel and L-gel group. Similarly, the enhanced antitumor effects were supported by significant inhibition of tumoral vasculature, confirmed by TUNEL assay and CD31 immunohistochemistry studies. Also, peritumorally injected PL gel group showed less toxicity to the other tissues as compared to that after oral LAPA administration and this effect was contributed to the reduced dose of LAPA for peritumoral injection. Hence, the formulated localized co-delivery of PTX and LAPA as PL-gel might be an excellent drug delivery alternative for the treatment of breast cancer with minimal toxicity to other organs of the body [51].
⦁ Liposomes through Nanochannel Device
Liposomes are one of the carrier systems rigorously investigated for the solubility enhancement of highly hydrophobic drugs, by virtue of their bilayered structure conferring them high entrapment potential of hydrophobic drugs even in an aqueous environment [59]. But its drawback is short biological half-life due to its quick clearance by RES [79]. This challenge can be encountered by PEGylation of liposomes which forbid them from recognition and uptake by RES, thereby extends the blood circulation time [59]. Further, the sustained release delivery of liposomes was achieved by metronomic chemotherapy, which involves a regime consisting of delivering small doses of a drug at a constant and short time intervals for prolonged treatments, mimicking the sustained delivery [80-82]. Based on this platform, Celia et al. [83] formulated the ultrastable lipid/cholesterol/PEG liposomes, loaded with LAPA, and then adopted an implantable nanochannel delivery system (nDS) for
subcutaneous implantation to provide sustained and continuous delivery of intact liposomes. High cholesterol content provided them greater rigidity. The prepared empty liposomes (E- Lip) and LAPA loaded liposomes (L-Lip) possessed spherical shape with 115 nm and 106 nm average diameter, respectively, with negative zeta potential, confirmed by dynamic light scattering and atomic force microscopy. Such a small size conferred them passive targeting ability through EPR effect. They can serve as a better carrier as they showed better drug encapsulation (62%) and loading (58 µg of drug per mg of lipids) efficiency.
The results of stability study showed that L-lipo is more stable than E-Lipo since the size and PDI of E-Lipo increases rapidly over the time. The constant net negative charge on Lipo suggested their high circulation half-life [84]. Further, DSC analysis of liposomes prepared at different LAPA to lipid ratio confirmed the interactions of LAPA with lipids, converting them in liquid crystalline phase, and indicated the retention of LAPA within liposomes. These results were in good agreement with drug retention investigation of liposomes at 37 ºC in PBS, 10% FBS/PBS (both pH 7.4) and PBS (pH 5.5) mimicking fresh liposomal solution, intestinal fluid, and tumoral environment, respectively. Lastly, the profound effect of nanochannel size on delivery of liposome from of nDS was investigated. The results demonstrated the release of liposomes followed zero order kinetics and exponential kinetics from 200 nm and 900 nm channels size nDS with a total release of 35% and 77%, respectively. Moreover, the liposomes were shown to intravasate into the systemic circulation after subcutaneous administration of liposomes and their internalization as well as anticancer activity on LAPA responsive BT474 BC cells were confirmed by employing confocal microscopy and proliferation assay. The characteristic time and dose-dependent reduction in cell proliferation were observed suggesting the retention of biological activity LAPA delivered by L-lipo and unaltered delivery of drug load by liposomes even after diffusion through nanochannels. The above-mentioned facts suggest the excellent potential of nDS to serve as a metronomic chemotherapeutic device, providing sustained delivery of hydrophobic anticancer drug, by exploiting the combination of principles of nanofluidics and nanotechnologies [83].
⦁ Lapatinib Conjugates for Diagnostic purpose
Not only the breast carcinoma but also various cancers like gastric, gallbladder, ovarian, cervical, uterine, testicular, and bladder cancer are characterized by overexpression of HER2 which can be employed as a prognostic biomarker for the detection and diagnosis of above-
said cancers [85,86]. On this basis, Gniazdowska et al. [87] postulated that the LAPA, via nanoparticles with a radiolabelled ligand, may be deployed as tumor imaging agent for BC. They synthesized the novel conjugate of LAPA with Technetium-99m (99mTc) (99mTc(NS3)(CN-LAPA) complex by conjugation, having high purity with specific activity within the range of 25-30GBq/µmol. Experiments were carried out to investigate their stability, and the results revealed that the conjugate had higher stability in the presence of the abundance of histidine and cysteine, since it didn’t undergo ligand interchange reaction. Further, their stability was confirmed in human serum showing resistance to enzymatic biodegradation. The optimum lipophilicity (logP ≈ 1.24) and low molecular weight (968.5g/mol) of a conjugate made it easy to distribute more efficiently in the body as contrary to trastuzumab (humanized IgG1 monoclonal antibody). The high affinity of the conjugate to HER2 receptor of SKOV-3 cells was evidenced by their saturable and specific binding to the receptor with low dissociation constant values, determined during saturation binding experiments on SKOV-3 cancer cell lines. Bio-distribution studies in mice implied the minimal uptake in all studied organs, however slightly higher uptake in liver and kidneys indicated that the conjugate follows the hepatic and renal clearance route. Moreover, the higher cost and the significant cardiac toxicity of trastuzumab can be avoided by employing LAPA conjugated radionuclide [71,88]. With the above mentioned benefits with easy synthesis and availability of 99mTc in hospital laboratory suggested the promising potential of the prepared conjugate as more effective diagnostic radiopharmaceutical and tumor imaging agent in HER2 positive breast cancer [87].
A similar attempt has been made by Wang et al. [89] for living cell membrane imaging. They synthesized water soluble conjugated polymer (WSCP), polythiophene (PT), containing a large number of chromic units in the backbone and hydrophilic groups in side chains to which LAPA was covalently linked for targeting intracellular domain of transmembrane proteins. Recently, WSCPs attracted the attention of cell imaging research arena due to their physicochemical properties like high quantum yield, tunable absorption and emission spectra, strong light harvesting ability and excellent photostability [90]. The prepared PTL had amphiphilic character by virtue of which they could form nanoscale aggregates with an average diameter of 13 nm in water conferring them easiness for endocytosis. These PTL aggregates reported having unaltered light harvesting ability and fluorescent properties as compared to that of its non-LAPA linked PT indicating an unaltered emissive property of the polymer even after covalent linking of LAPA. Further, results of MTT cell cytotoxicity on
cancer cell lines (e.g. MCF-7, SKBr-3 and MDA-MB-23), revealed the slight toxicity of PTL on MCF-7 whereas no toxicity on other two cell lines under study for five days incubation period. Owing to their lower toxicity to cell lines, PTL can be used for cell imaging. Further, confocal laser scanning microscopy indicated the localization of PTL in the cell membrane region and achieved cell membrane specific imaging, in contrast to cytoplasmic localization of other previously reported PT derivatives. Thus, all these facts, regarding WSCPs, e.g. PTL, proved their potential as targeted imaging aid for cell membrane imaging and cancer diagnosis [89].
⦁ Conclusion
Nowadays, breast cancer became one of the leading causes of death in women. The principles of drug targeting were successfully exploited to design new drugs like Lapatinib, a selective inhibitor of EGFR and HER2 which are expressed by cancer cells to avoid toxicities to other organs of the body. Various nanoformulations were investigated and reported as better DDS for LAPA as compared to Tykerb® (an unique marketed dosage form) since LAPA encapsulation in nanoformulations indicated to enhance its aqueous solubility, in turn, the dissolution and bioavailability of the drug, avoid drug contact with plasma proteins, and increases circulation half-life. Also, nanoformulations, through their active and passive targeting potential to tumor tissues, demonstrated enhanced delivery amount to the intended site. These nanofomulations included nanoparticles, nanocapsules, polymeric micelle, nanochannel DDS etc. whereas some research groups also reported the successful delivery of lapatinib via dosage forms like liposomes. Conclusively, nanoformulation approach could serve as a promising delivery vehicle for LAPA.
⦁ Expert opinion:
Development of LAPA DDS is less explored topic for BC treatment. The facts discussed in the present review not only thrown light on the developed DDS beyond oral route of administration but also provided sufficient evidence showing their greater potential for treatment of BC. LAPA nano-DDS demonstrated superiority over the oral tablet by attaining higher bioavailability through targeting and by surface charge modification. Further, this approach minimized the toxicity to normal tissues by reduction in dose size. In spite of such advantages offered by multiparticulate and nano-delivery systems, their clinical application is
limited due to pharmaceutical problems, e.g. their stability, difficulty in scale-up, technology transfer to commercial scale etc. Also, pharmaceutical manufacturing industries focus their research around cheap and easy to manufacture conventional dosage forms.
Till date, Tykerb® is the unique marketed tablet dosage form of LAPA for oral administration which needs to be taken at very high dose. Therefore, pharmaceutical industries should pay special attention towards the development of alternative novel dosage forms like nanoparticles, micelle, and liposomes etc. Also, other non-oral routes of administration should also be explored for efficient delivery of LAPA to the tumor tissue.
Rather treatment with single chemotherapeutic agents, a combination of LAPA with other drugs could offer more effective management of BC. Various previous clinical trials proved the better efficiency of its combination with PTX, capecitabine, letrozole for different types of BC. As discussed in the review, few research groups attempted combination therapy of LAPA and PTX only. Other combination should also be tried for development of DDS. Further, their clinical trials should also be promoted by the clinicians and physicians.
The novel DDS, developed through laboratory scale research, are in vain unless it is commercialized and made available in the market for the appropriate patient. The costlier technologies employed in manufacturing and their evaluations are making products more costly. At the same time, the cost should be minimized so that it is affordable by patients without compromising its therapeutic efficacy. This critical balance should be achieved by designing simple and cost effective unit operations for formulation and modifying existing manufacturing skills for cost effective operations. Pharmaceutical and industrial manufacturing experts should pay special attention towards refinement and modification of scale up technologies and their transfer to manufacturing scale, in view of more economical perspective. Rather than above facts, health and socioeconomic hazards are some unavoidable risks in the employment of nanotechnology in pharmaceutical practice. Therefore, scientists from various fields related to pharmacy, clinicians and health specialists especially oncologists should work together through an interdisciplinary approach to explore and maximize the treatment potential of LAPA to the appropriate patients at affordable cost.
Acknowledgement:
The authors are thankful to Ministry of Human Resources and Development, New Delhi (India) and Department of Pharmaceutical Engineering & Technology, IIT (BHU), Varanasi (U.P., India) for providing financial assistance.
Conflict of interest:
There are no conflicts of interest.
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Table 1 Drug summary.
S. No. Parameter Description
1. Name Lapatinib
2. Alternate Name Lapatinib ditosylate, Tyverb®, Tykerb®
3. Chemical Formula C29H26ClFN4O4S
4. Structure
5. Half-life 14.2 hours: After single dose administration
24 hours : After multiple effective dose
6. LogP 5.4
7. Average Molecular
Weight 581.058
8. Clinical indications Metastatic breast cancer, postmenopausal women
with hormone receptor positive metastatic cancer, hormone receptor-negative metastatic cancer
9. Mechanism of Action ⦁ It is a small molecular member of the 4- anilinoquinazoline family of kinase inhibitors.
⦁ It targets the intracellular tyrosine kinase domains of the both receptors, HER2 and EGFR
⦁ It seems to interrupt cell proliferation and tumor
growth via inhibition of ErbB-driven cell growth
Table 2 Summary of drug delivery systems for lapatinib.
S.
No. Author Nanocarrier Polymer/s used Indication
1. Wan et al. [61, 62] Nanoparticles Human Serum Albumin and Egg
Yolk Lecithin Triple negative Breast Cancer and HER2ǂ
positive Breast Cancer
2. Huo et al.[56] Nanoparticles PLGA-TPGS* and DSPE-PEG# (for
encapsulation) Breast Cancer
3. Mobasseri et
al. [66] Nanoparticles Dextran sulphate and
chitosan HER2 positive Breast
Cancer
4. Zhang et al.
[67] Nanoparticles Bovine serum albumin
and Egg Yolk Lecithin Breast Cancer
5. Gao et al.
[29, 60] Core-shell
Nanoparticles Bovine serum albumin
and Egg Yolk Lecithin Glioma
6. Vergara et al.
[48] Nanocapsules Alginic acid and
Chitosan Multidrug resistant
ovarian cancer
7. Wang et al.
[40] Polymeric Micelle PEG-PBC$ Multidrug resistant breast
cancer
8. Wei et al. [49] Polymeric Micelle LAPA conjugated
PEG-PLA¥ HER2 positive Breast
Cancer
9. Li et al. [47] Polymeric Micelle PEG-PCDY Prostate Cancer
10. Dehghan et
al. [50] Polymeric Micelle Pluronic F127 Metastatic breast cancer
11. Hu et al.
[51] Microparticles via
In Situ Gel Pluronic F127 HER2 and P-gp positive
breast cancer
12. Celia et al.
[83] Liposomes via
nanochannel device PCS100/Chol/DSPE-
MPEG 2000## Breast Cancer
13. Gniazdowska
et al. [87] LAPA conjugate (99mTc(NS3)(CN-
LAPA)** complex HER2 positive Breast
Cancer
14. Wang et al.
[89] LAPA conjugate LAPA-polytiophene HER2 and EGFR†
expressing cancers
*: poly[lactide-co-glycolide]-D-a-tocopheryl polyethylene glycol 1000 succinate; #:1,2- distearoyl -sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000; $: Poly(ethylene glycol)-block-poly (2-methyl-2-benzoxycarbonylpropylene carbonate; ¥: Poly(ethylene glycol)-block -poly(lactic acid); Y: Poly(ethylene glycol)-block-poly(2-methyl-2- carboxyl-propylene carbonate-graft-dodecanol; ##: egg L-α-phosphatidylcholine /cholesterol/ N- (carbonyl-methoxy polyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3- phosphoethanolamine; **:technetium-99m-(tris(2-mercaptoethyl)-amine;2,20,200- nitrilotriethanethiol)- isocyanobutyric acid succinimidyl ester-Lapatinib; ǂ:Human epidermal growth factor receptor; †: Epidermal growth factor receptor.
Table 3 Characteristics of nanocarrier systems and purpose to use for drug delivery.
S. No. Characteristics Purpose
1. Size (approx. upto 200-250
nm) For passive targeting via enhanced permeation and
retention effect [49, 54, 55]
2. Surface Charge induction For higher uptake in cancer cell [56], to escape from RES* [57] and for prolonging systemic
circulation time [59]
3. Surface engineering with
ligands or antibodies For active targeting [58] and minimize side effects
to un-intended organs [49]
4. Core-Shell Structure especially in nanocapsules, micelle and some
Nanoparticles For achieving higher drug loading and enhance solubility of hydrophobic drugs particularly BCS# Class II drugs [29]
5. Sustained or controlled delivery For minimizing total drug dose [60], increasing
therapeutic efficacy with less side effects of drug [51]
6. Encapsulation of drug For avoiding systemic drug loss due to first pass metabolism and plasma protein binding leading to
enhanced bioavailability [56]
* Reticuloendothelial system ; # Biopharmaceutical Classification System
Article highlights
⦁ Lapatinib is a targeted dual tyrosine kinase inhibitor of EGFR and HER2, overexpressed by tumor cells of breast cancer.
⦁ Its clinical use is limited due to its poor aqueous solubility, poor bioavailability, high binding affinity towards blood proteins, and toxicities related to its higher dose. Also, stability and scale-up issues also contribute to the commercial availability of nano-delivery systems
⦁ Nano-delivery system could serve as a promising alternative by virtue of their benefits like easy surface modification for active targeting and longer circulation half-life, passive targeting via enhanced permeation and retention effect.
⦁ Various nano-delivery systems, including nanoparticles, polymeric micelle, core- shell nanoparticles, nanochannel, etc., were investigated for lapatinib delivery and summarized in the present review.
⦁ On the other hand, owing to its exclusive selectivity to above-said receptors, they are employed as diagnostic agents for detection of breast cancer through its conjugate with imaging agents.
⦁ Lapatinib also reported to have multidrug resistant reversal property, which could be exploited to treat resistant cancer by its novel combination with paclitaxel.
⦁ It could be suggested that, the scale-up and technology transfer techniques should
be revised and modified, in light of manufacturing of nano-delivery systems of lapatinib, for commercial level production.
This box summarizes key points contained in the article.
List of Abbreviations
BC : Breast cancer
CI50 : Combination index at 50% cell growth inhibition CMC : Critical micelle concentration
CSC : Cancer stem cells
CSN : Core-shell nanoparticles DDS : Drug delivery system DOX : Docetaxel
EE : Encapsulation efficiency
EGFR : Epidermal growth factor receptor E-Lip : Empty liposomes
EPR : Enhanced permeation and retention ER : Estrogen receptor
HER2 : Human epidermal growth factor receptor HSA : Human serum albumin
IC50 : Inhibitory concentration for 50% cell growth inhibition IgG : Immunoglobulin G
Kd : Dissociation constant LAPA : Lapatinib
L-Lip : Lapatinib loaded liposomes LNs : Lapa nanoparticles
LS : Lapatinib solution
LTNP : Lipoprotein like nanoparticles MDR : Multidrug resistant
MPs : Microparticles
MTT : 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide nDS : Nanochannel delivery system
NPs : Nanoparticles
PBS : Phosphate buffer saline PDI : Polydispersivity index PEG : Polyethylene glycol PF127 : Pluronic F127
PLPT : Polymeric lipid hybrid nanoparticles PM : Polymeric micelle
PM-LP : Lapatinib polymeric micelle PR : Progesterone receptor
PT : Polythiophene
PTX : Paclitaxel
RES : Reticuloendothelial system SEM : Scanning electron microscopy
SLBL : Sonication-assisted layer-by-layer TEM : Transmission electron microscopy TKI : Tyrosine kinase inhibitor
TNBC : Triple negative breast cancer WSCP : Water soluble conjugated polymer
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