Pegylated liposomal doxorubicin: a review of its use in metastatic breast cancer, ovarian cancer, multiple myeloma and AIDS-related Kaposi's sarcoma

Pegylated liposomal doxorubicin (Caelyx?, Doxil?) represents an improved formulation of conventional doxorubicin, with reduced cardiotoxicity and an improved pharmacokinetic profile. This article reviews the efficacy and tolerability of pegylated liposomal doxorubicin in metastatic breast cancer, progressive ovarian cancer, relapsed or refractory multiple myeloma and AIDS-related Kaposi's sarcoma, as well as summarizing its pharmacological properties. In three randomized, open-label, multicentre trials, monotherapy with pegylated liposomal doxorubicin was as effective as doxorubicin or capecitabine in the first-line treatment of metastatic breast cancer, and as effective as vinorelbine or combination mitomycin plus vinblastine in taxane-refractory metastatic breast cancer. Pegylated liposomal doxorubicin alone was as effective as topotecan or gemcitabine alone in patients with progressive ovarian cancer resistant or refractory to platinum- or paclitaxel-based therapy, according to the results of three randomized multicentre trials. In addition, in patients with progressive ovarian cancer who had received prior platinum-based therapy, progression-free survival was significantly longer with pegylated liposomal doxorubicin plus carboplatin than with paclitaxel plus carboplatin, according to the results of a randomized, open-label multicentre trial. Combination therapy with pegylated liposomal doxorubicin plus bortezomib was more effective than bortezomib alone in patients with relapsed or refractory multiple myeloma, according to the results of a randomized, open-label, multinational trial. Randomized multinational trials also demonstrated the efficacy of pegylated liposomal doxorubicin in patients with advanced AIDS-related Kaposi's sarcoma. Pegylated liposomal doxorubicin exhibited a relatively favourable safety profile compared with conventional doxorubicin and other available chemotherapy agents. The most common treatment-related adverse events included myelosuppression, palmar-plantar erythrodysesthesia and stomatitis, although these are manageable with appropriate supportive measures. To conclude, pegylated liposomal doxorubicin is a useful option in the treatment of various malignancies, including metastatic breast cancer, ovarian cancer, multiple myeloma and AIDS-related Kaposi's sarcoma.     

Pegylated liposomal doxorubicin in the treatment of cancers of the breast and ovary

Peglyated liposomal doxorubicin was developed to maintain or enhance the demonstrated antineoplastic effects of doxorubicin, while improving the toxicity profile associated with this important cytotoxic agent. Accumulating clinical data have confirmed the activity of pegylated liposomal doxorubicin in cancers of the breast and ovary. Furthermore, Phase II and III trial experience has revealed that the drug produces objective responses comparable in rate and duration to doxorubicin and other single agents employed in metastatic breast cancer. In recurrent and platinum-resistant ovarian cancer, single-agent pegylated liposomal doxorubicin has assumed an important role in routine patient management.     

Pegylated liposomal doxorubicin in the treatment of cancers of the breast and ovary

Peglyated liposomal doxorubicin was developed to maintain or enhance the demonstrated antineoplastic effects of doxorubicin, while improving the toxicity profile associated with this important cytotoxic agent. Accumulating clinical data have confirmed the activity of pegylated liposomal doxorubicin in cancers of the breast and ovary. Furthermore, Phase II and III trial experience has revealed that the drug produces objective responses comparable in rate and duration to doxorubicin and other single agents employed in metastatic breast cancer. In recurrent and platinum-resistant ovarian cancer, single-agent pegylated liposomal doxorubicin has assumed an important role in routine patient management.     

Doxorubicin liposomal pegylated: new preparation. Breast cancer: not just a question of short-term cardiac effects

(1) There is no reference first-line chemotherapy regimen for metastatic breast cancer. Anthracycline-based combinations are generally used. One of the main problems with anthracyclines is the risk of heart failure, both during and some time after treatment. (2) A liposomal pegylated doxorubicin, an anthracycline, is now available in Europe. The aim of pegylation is supposedly to reduce the cardiotoxicity relative to standard doxorubicin. The marketing licence specifies that liposomal pegylated doxorubicin must not be used in combination with other drugs in people with metastatic breast cancer. This is the second liposomal doxorubicin preparation to be authorised for this use in France; we concluded that the first product, a non-pegylated form, offered no therapeutic advance. (3) According to the only available comparative trial, liposomal pegylated doxorubicin is no more effective than standard doxorubicin in terms of the duration or quality of survival. (4) In this trial, liposomal pegylated doxorubicin was associated with slightly fewer cardioechographic abnormalities than standard doxorubicin. (5) Other adverse events were also less common (hair loss, nausea and vomiting, and neutropenia), while some were more common (palmoplantar erythrodysesthesia, stomatitis and mucitis). Overall, 24% of patients stopped using liposomal pegylated doxorubicin because of adverse events, compared with 11% of patients receiving standard doxorubicin. (6) Unlike liposomal non-pegylated doxorubicin, the liposomal pegylated form is no more difficult than standard doxorubicin to prepare for injection. (7) In practice, when the decision is made to use doxorubicin, the standard form, at an appropriate dose, is suitable for most patients, as long as cardiac function is closely monitored. Differences in the adverse effect profile (especially hair loss) may make liposomal pegylated doxorubicin more attractive to some patients (it costs 20 times more than standard doxorubicin in France).     

A pegylated liposomal platform: pharmacokinetics, pharmacodynamics, and toxicity in mice using doxorubicin as a model drug

Aims were to observe pharmacokinetics, pharmacodynamics, and toxicity for constructing a Sino-pegylated liposomal platform. Human hepatocarcinoma cells (Bel7402) and murine hepatocarcinoma cells (H(22)) were used for the cytotoxicity assay and the in vivo solid xenograft tumor model in mice, respectively. Pharmacokinetic results in mice showed that the pegylated liposomal doxorubicin markedly prolonged the blood circulation of doxorubicin. Elimination half-time (T(1/2,gamma)) of pegylated, regular liposomal doxorubicin and free doxorubicin were 46.09 +/- 14.44, 26.04 +/- 3.34, and 23.72 +/- 5.13 h, respectively. The area under the concentration-time curves (AUC(0- infinity )) (h. microg/g) of the pegylated and regular liposomal doxorubicin were 6.8- and 2.6-fold higher than that of free doxorubicin, respectively. Cytotoxicity and antitumor activity in vivo indicated that activity of the pegylated liposomal doxorubicin was higher than that of the regular or the free one, respectively. After two weeks of tail intravenous injection of the pegylated liposomal doxorubicin at a single dose of 10 mg/kg, no significant damage was observed in gastric, intestinal mucosa, and heart muscle, but pronounced damages were found in the control group after dosing free doxorubicin. The results demonstrate that the pegylated liposomes improve the efficacy of toxics and reduce the toxicity, therefore providing favorable evidence for building a pegylated liposomal platform.     

Pharmacokinetics of pegylated liposomal Doxorubicin: review of animal and human studies

Pegylated liposomal doxorubicin (doxorubicin HCl liposome injection; Doxil or Caelyx) is a liposomal formulation of doxorubicin, reducing uptake by the reticulo-endothelial system due to the attachment of polyethylene glycol polymers to a lipid anchor and stably retaining drug as a result of liposomal entrapment via an ammonium sulfate chemical gradient. These features result in a pharmacokinetic profile characterised by an extended circulation time and a reduced volume of distribution, thereby promoting tumour uptake. Preclinical studies demonstrated one- or two-phase plasma concentration-time profiles. Most of the drug is cleared with an elimination half-life of 20-30 hours. The volume of distribution is close to the blood volume, and the area under the concentration-time curve (AUC) is increased at least 60-fold compared with free doxorubicin. Studies of tissue distribution indicated preferential accumulation into various implanted tumours and human tumour xenografts, with an enhancement of drug concentrations in the tumour when compared with free drug. Clinical studies of pegylated liposomal doxorubicin in humans have included patients with AIDS-related Kaposi's sarcoma (ARKS) and with a variety of solid tumours, including ovarian, breast and prostate carcinomas. The pharmacokinetic profile in humans at doses between 10 and 80 mg/m(2) is similar to that in animals, with one or two distribution phases: an initial phase with a half-life of 1-3 hours and a second phase with a half-life of 30-90 hours. The AUC after a dose of 50 mg/m(2) is approximately 300-fold greater than that with free drug. Clearance and volume of distribution are drastically reduced (at least 250-fold and 60-fold, respectively). Preliminary observations indicate that utilising the distinct pharmacokinetic parameters of pegylated liposomal doxorubicin in dose scheduling is an attractive possibility. In agreement with the preclinical findings, the ability of pegylated liposomes to extravasate through the leaky vasculature of tumours, as well as their extended circulation time, results in enhanced delivery of liposomal drug and/or radiotracers to the tumour site in cancer patients. There is evidence of selective tumour uptake in malignant effusions, ARKS skin lesions and a variety of solid tumours. The toxicity profile of pegylated liposomal doxorubicin is characterised by dose-limiting mucosal and cutaneous toxicities, mild myelosuppression, decreased cardiotoxicity compared with free doxorubicin and minimal alopecia. The mucocutaneous toxicities are dose-limiting per injection; however, the reduced cardiotoxicity allows a larger cumulative dose than that acceptable for free doxorubicin. Thus, pegylated liposomal doxorubicin represents a new class of chemotherapy delivery system that may significantly improve the therapeutic index of doxorubicin.     

Repeated injection of pegylated liposomal antitumour drugs induces the disappearance of the rapid distribution phase

Upon repeated administration, empty pegylated liposomes lose their long‐circulating characteristics, referred to as the accelerated blood clearance (ABC) phenomenon. To investigate whether cytotoxic drug‐containing pegylated liposomes could also elicit a similar phenomenon, two pegylated liposomal antitumour drugs (doxorubicin and mitoxantrone) were prepared, and they were administrated twice in the same animals with a 10‐day interval at a dose level of 8 mg kg^?1 (pegylated liposomal doxorubicin) and 4 mg kg^?1 (pegylated liposomal mitoxantrone). By comparing the overall pharmacokinetics after a single‐dose injection with that in animals treated with two doses, it was surprising to find that repeated administration of pegylated liposomal antitumour drugs caused the disappearance of rapid distribution phase instead of the ABC phenomenon, resulting in the conversion of a two‐compartment model to a one‐compartment model. Further investigation revealed that repeated injection induced the decreased uptake of liposomal antitumour drugs by the spleen at the early time point of 0.5–8 h after injection. In contrast, the deposition of liposomal antitumour drugs into liver was not affected. Therefore, the disappearance of the rapid distribution phase might be related to the reduced spleen uptake at the early time point.     

聚乙二醇多柔比星脂质体及表柔比星在乳腺癌新辅助化疗中的应用比较

目的 比较聚乙二醇多柔比星脂质体及表柔比星在乳腺癌新辅助化疗中的疗效及不良反应。方法 回顾性分析2015年1月-12月行乳腺癌AC-T或EC-T新辅助化疗的146例患者,其中应用聚乙二醇多柔比星脂质体86例,应用表柔比星60例,分析其临床病理特征、化疗效果及不良反应,并应用Logistic回归分析影响新辅助化疗疗效的因素。结果 聚乙二醇多柔比星脂质体组与表柔比星组临床病理特征一致,无统计学差异。2组新辅助化疗的疗效无统计学差异。HER-2状态是影响新辅助化疗疗效的主要因素。聚乙二醇多柔比星脂质体组的白细胞降低、消化道反应、脱发及心电图异常等不良反应较表柔比星组明显减少,而手足综合征则明显增多,具有统计学差异。结论 聚乙二醇多柔比星脂质体与传统的表柔比星在乳腺癌新辅助化疗中疗效一致。     

Allometric scaling of pegylated liposomal anticancer drugs

Pegylated liposomal formulations contain lipid conjugated to polyethylene glycol. The disposition of encapsulated drug is dictated by the composition of the liposome, thus altering the pharmacokinetic (PK) profile of the drug. Allometric scaling is based on a power-log relationship between body weight (W) and drug clearance (CL) among mammals and has been used to compare the disposition of nonliposomal drugs across species. The objectives of this study were to use allometric scaling to: (1) compare the disposition of pegylated liposomal drugs across speciesand determine the best scaling model and (2) predict PK parameters of pegylated liposomal drugs in humans. The PK of pegylated liposomal CKD-602 (S-CKD602), doxorubicin (Doxil^®), and cisplatin (SPI-077) were compared. PK studies ofS-CKD602, Doxil^®, and SPI-077 were performed at the maximum tolerated dose (MTD) in male and female mice, rats, dogs and patients with refractory solid tumors. The allometric equation used to evaluate the relationship between W and CL in each species was CL = a(W)^m (a = empirical coefficient; m = allometric exponent). Substitution of physiological variables other than body weight, such as factors representative of the mononuclear phagocyte system (MPS) were evaluated. Dedrick Plots and Maximum Life-Span Potential (MLP) were used to determine scaling feasibility. Standard allometry demonstrated a relationship between clearance of S-CKD602, Doxil^®, and SPI-077 and body, spleen, liver, and kidney weights, total monocyte count, and spleen and liver blood flow. However, using scaling to predict CL of these agents in humans often resulted in differences >30%. Despite a strong correlation between body weight and MPS-associated variables with CL among preclinical species, the use of the equations did not predict CL. Thus, new methods of allometric scaling and measures of MPS function need to be developed.     

Investigation of the Discriminatory Ability of Pharmacokinetic Metrics for the Bioequivalence Assessment of PEGylated Liposomal Doxorubicin

Purpose

The purpose of the study was to construct a population pharmacokinetic model for pegylated liposomal doxorubicin and use the final model to investigate the discrimination performance of pharmacokinetic metrics (e.g., C_max, AUC and partial AUC) of various analytes (e.g., liposome encapsulated doxorubicin, free doxorubicin and total doxorubicin) for the identification of formulation differences by means of Monte Carlo simulations.

Methods

A model was simultaneously built to characterize the concentration time profiles of liposome-encapsulated doxorubicin and free doxorubicin using NONMEM. The different scenarios associated with changes in release rate (Rel) were simulated based on the final parameters. 500 simulated virtual bioequivalence (BE) studies were performed for each scenario, and power curves for the probability of declaring BE were also computed.

Results

The concentration time profiles of liposome-encapsulated doxorubicin and free doxorubicin were well described by a one- and two-compartment model, respectively. pAUC_0-24 h and pAUC_0-48 h of free doxorubicin was most responsive to changes in the Rel when the Rel (test)/Rel (reference) ratios decreased. In contrast, when the Rel (test) increased, AUC_0-t of liposome-encapsulated doxorubicin was the most responsive metric.

Conclusions

In addition to the traditional metrics, partial AUC should be included for the BE assessment of pegylated liposomal doxorubicin.

     

Liposomal delivery of photosensitising agents

Photodynamic therapy is a clinically approved treatment for cancer and noncancer diseases. This modality utilises light-activatable chemicals (photosensitising agents) to capture photons and use light energy for the production of cytotoxic reactive molecular species. Most photosensitisers that are in use clinically or in preclinical development are hydrophobic and tend to aggregate in the aqueous environment, which limits their delivery and photosensitising efficiency. Liposomal delivery of photosensitisers will often overcome or decrease these problems. In addition, as with chemotherapeutic agents, liposomal formulations of photo-sensitising agents may help to achieve better selectivity for tumour tissue compared with normal tissue. Over the past years, liposomal photosensitisers have emerged as therapeutic agents in many experimental studies, and have obtained approval for clinical applications. Recent progress in liposomal technology further opens up the possibility of generating more selectively targeted photosensitisers encapsulated in liposomes. This review will cover progress in the use of liposomal photosensitisers, summarise current liposomal formulations, and project future directions for the liposomal delivery of photosensitising agents.     

Liposomal delivery of photosensitising agents

Photodynamic therapy is a clinically approved treatment for cancer and noncancer diseases. This modality utilises light-activatable chemicals (photosensitising agents) to capture photons and use light energy for the production of cytotoxic reactive molecular species. Most photosensitisers that are in use clinically or in preclinical development are hydrophobic and tend to aggregate in the aqueous environment, which limits their delivery and photosensitising efficiency. Liposomal delivery of photosensitisers will often overcome or decrease these problems. In addition, as with chemotherapeutic agents, liposomal formulations of photo-sensitising agents may help to achieve better selectivity for tumour tissue compared with normal tissue. Over the past years, liposomal photosensitisers have emerged as therapeutic agents in many experimental studies, and have obtained approval for clinical applications. Recent progress in liposomal technology further opens up the possibility of generating more selectively targeted photosensitisers encapsulated in liposomes. This review will cover progress in the use of liposomal photosensitisers, summarise current liposomal formulations, and project future directions for the liposomal delivery of photosensitising agents.     

Management strategies for recurrent platinum-resistant ovarian cancer

Although ovarian cancer is often a chemosensitive malignancy, patients who are resistant to platinum-based chemotherapy represent a therapeutic challenge. Currently, the only drugs that are US FDA approved to treat this subset of patients are paclitaxel, pegylated liposomal doxorubicin (PLD) and topotecan. The response rates with these agents is in the 10-15% range and overall survival is around 12 months. Other drugs that have shown some activity in platinum-resistant ovarian cancer include the taxane analogues, oral etoposide, pemetrexed and bevacizumab. Unfortunately, randomized phase III trials of second-line chemotherapy in patients with platinum-resistant ovarian cancer have not shown an advantage over existing therapy with respect to progression-free survival or overall survival. The only trial that has reported a significant progression-free survival advantage over standard therapy is a randomized phase II trial of PLD with or without EC145, a folate-linked vinca alkaloid. Final survival results of this trial are pending.     

Role of pegylated liposomal doxorubicin (Caelyx) in the treatment of relapsing ovarian cancer

The most significant factor predicting response to second-line chemotherapy is the time interval elapsed from the end of chemotherapy to relapse occurrence. Two types of situations may be considered: patients with platinum-sensitive relapse (relapse-free interval longer than 6 months) and patients with platinum-refractory relapse (progression during treatment or relapse-free interval under 6 months). Pegylated liposomal doxorubicin is a doxorubicin formulation. Encapsulation in liposomes confers it different pharmacokinetic characteristics and a more favorable toxicity profile. The following review examines the efficacy and safety of pegylated liposomal doxorubicin for the treatment of relapsing epithelial ovarian cancer.     

聚乙二醇多柔比星脂质体联合化疗治疗晚期恶性肿瘤临床观察

目的探讨以聚乙二醇多柔比星脂质体为主联合化疗治疗晚期恶性肿瘤的临床疗效和不良反应。方法收集2007年1月—2009年11月以聚乙二醇多柔比星脂质体为主的联合化疗治疗晚期恶性肿瘤患者的治疗信息、临床情况,并对患者进行跟踪随访至2010年4月30日。结果 33例恶性肿瘤患者共计完成化疗106周期,中位3周期（1~8周期）,可评价疗效28例,其中完全缓解4例,占14.3%;部分缓解11例,占39.3%;稳定11例,占39.3%;进展2例,占7.1%;客观有效率为53.6%,临床受益率为92.9%。随访周期6~39个月,中位随访时间16个月,总生存时间4~39个月,中位生存时间16个月,无疾病进展时间2~39个月,中位无疾病进展时间为10个月,1和2年生存率分别为69.2%和34.2%,3例生存3年以上。不良反应主要为骨髓抑制、脱发和胃肠道反应,另外乏力、手足综合症常见,心脏毒性轻微。结论以聚乙二醇多柔比星脂质体为主联合化疗治疗晚期恶性肿瘤是有效的,患者可以良好耐受。     

A comparison of liposomal formulations of doxorubicin with drug administered in free form: changing toxicity profiles.

The anthracycline antibiotic doxorubicin has wide activity against a number of human neoplasms and is used extensively both as a single agent and in combination regimens. In addition to the use of free, unencapsulated doxorubicin, there are two US Food and Drug Administration approved liposomal formulations of doxorubicin currently available, with several additional liposomal formulations being researched either in the laboratory or in clinical trials. The two approved liposomal formulations of doxorubicin have significantly different lipid compositions and loading techniques, which lead to both unique pharmacokinetic and toxicity profiles, distinct from those of the unencapsulated form. This article discusses the toxicities associated with the free form of doxorubicin, as well as those associated with the two most common liposomal formulations, namely Doxil and Myocet. One of the key toxicity issues linked to the use of free doxorubicin is that of both an acute and a chronic form of cardiomyopathy. This is circumvented by the use of liposomal formulations, as these systems tend to sequester the drug away from organs such as the heart, with greater accumulation in liver, spleen and tumours. However, as will be discussed, the liposomal formulations of doxorubicin are not without their own related toxicities, and, in the case of Doxil, may be associated with the unique toxicity of palmar-plantar erythrodysaesthesia. Overall, the use of liposomal doxorubicin allows for a greater lifetime cumulative dose of doxorubicin to be administered, however acute maximal tolerated doses differ significantly, with that of Myocet being essentially equivalent to free doxorubicin, while higher doses of Doxil may be safely administered. This review highlights the differences in both toxicity and pharmacokinetic properties between free doxorubicin and the different liposomal formulations, as have been determined in pre-clinical and clinical testing against a number of different human neoplasms. The need for further testing of the liposomal formulations prior to the replacement of free doxorubicin with liposomal doxorubicin in any established combination therapy regimens, as well as in combination with the newer therapeutics such as monoclonal antibodies is also discussed.     

脂质体多柔比星对初治多发性骨髓瘤疗效及安全性评估

目的：旨在分析脂质体多柔比星替代传统蒽环类药物多柔比星治疗多发性骨髓瘤（ MM）患者的有效率及安全性。方法收集2011年9月—2014年1月,共计45例初治MM患者,其中22例接受DVDT方案化疗（脂质体多柔比星20 mg· m－2· dL－1、长春新碱2 mg· d－1、地塞米松40 mg· d1－1～4、沙利度胺100 mg· d1－1－28）,23例接受VADT方案化疗（多柔比星10 mg· d1－1～4、长春新碱0．5 mg· d1－1～4、地塞米松40 mg· d1－1～4、沙利度胺100 mg· d1～28）;并对患者进行跟踪随访至2014年3月;评估分析化疗有效率及副反应。结果 DVDT组和VADT组的总体有效率相似（81．8％比73．8％,P＝0．780）,DVDT组骨髓抑制及心脏毒副反应较VADT组低,耐受性较好。结论脂质体多柔比星用于初治多发性骨髓瘤患者治疗的有效率和安全性均较好,适合免疫力低下且伴心脏功能不全的MM患者。     

Liposomal doxorubicin and weekly paclitaxel in the treatment of metastatic breast cancer

The combination of paclitaxel and doxorubicin or epirubicin is highly active against metastatic breast cancer, yet may produce congestive heart failure. Liposome-encapsulated doxorubicin is a new formulation of doxorubicin with no dose-limiting cardiac toxicity. Twenty-one patients with metastatic breast cancer were treated with pegylated liposomal doxorubicin (20 mg/m2, day 1) and paclitaxel (100 mg/m2, days 1 and 8) for six cycles every 2 weeks. All patients had had relapse or progression on one to five previous chemotherapies. We observed two patients with complete and eight patients with partial remissions (48% response rate). Eight of the 10 responders had had previous therapy with epirubicin, doxorubicin or mitoxantrone. The mean remission duration was 5 months. Disease progression due to brain metastasis occurred in five cases. Severe side effects (grade 3 WHO) were alopecia (100%), skin toxicity in 29%, neuropathy in 24% and mucositis in 13%. Leukopenia (grade 4 WHO) was observed in 48%, but there was no cardiac toxicity, no death and no hospitalization. The combination of weekly paclitaxel and liposomal doxorubicin every 2 weeks is highly effective in previously treated patients. Based on the doses we administered, we recommend 15 mg/m2 liposomal doxorubicin every 2 weeks and 80 mg/m2 paclitaxel weekly.     

Liposome encapsulation reduces cantharidin toxicity

Several reports have demonstrated that cantharidin is a strong anticancer compound in vitro; however, its in vivo usefulness is often limited due to its high systemic toxicity. In this study, we encapsulated cantharidin into pegylated liposomes and studied its activity against human breast cancer MCF-7 cells in vitro and its systemic toxicity in mice. Another two methods were also used to reduce the dosage of cantharidin, including labeling liposomal cantharidin with octreotide and exposing cells to hyperbaric oxygen. The cytotoxic activity of pegylated liposomal cantharidin was drastically reduced compared with free cantharidin in vitro. Octreotide-labeled pegylated liposomal cantharidin induced cell death by specifically targeting somatostatin receptors in MCF-7 cells. Cell death was augmented with a low dose of cantharidin under hyperbaric oxygen. Liposomal cantharidin had significantly less systemic toxicity than free cantharidin in vivo and also exhibited a high efficacy against antitumor growth in nude mice. These results suggest that the systemic toxicity of cantharidin can be mitigated by liposome encapsulation; however, that did not decrease its antitumor activity.