A pilot study investigating plasma pharmacokinetics and tolerance of oral capecitabine in carcinoma-bearing dogs.

BACKGROUND: Capecitabine is an oral prodrug of the active metabolite 5-fluorouracil, which has been used effectively in human colorectal, head and neck, and mammary carcinomas. Capecitabine has several properties that make it an attractive treatment option for dogs: (i) it is relatively inexpensive, (ii) it has a short half-life in humans, allowing for rapid plasma concentration changes to be achieved with dosage adjustments, (iii) it is effective for treating carcinomas in humans, for which there are no widely-effective oral chemotherapy options in dogs, and (iv) it is thought to preferentially target cancer cells due to different expression of thymidine phosphorylase, thereby decreasing the risk of off-target side effects. However, capecitabine has not been widely explored as a chemotherapy agent for dogs. The goal of this study was to determine the plasma disposition of capecitabine in dogs following a single oral dose and to document any adverse events associated with capecitabine administration over the course of 5 weeks. RESULTS: Capecitabine was well tolerated throughout the 5-week study period when administered to 5 dogs with naturally occurring carcinomas at 750 mg/m[Formula: see text] by mouth once daily for 14 consecutive days in a 3-week cycle. No dogs withdrew from the study due to adverse events or other causes. The median AUC[Formula: see text] was 890 h[Formula: see text]ng/ml (range 750-1100 h[Formula: see text]ng/ml); however, the maximum blood concentration and time to reach that concentration of capecitabine was highly variable after a single dose. CONCLUSIONS: Capecitabine appears well-tolerated as an oral chemotherapy agent for dogs with carcinomas, although individualized dosing may be necessary, and further studies are warranted.

Development of prednisone resistance in naïve canine lymphoma: Longitudinal evaluation of NR3C1α, ABCB1, and 11ß-HSD mRNA expression.

Prednisone resistance develops rapidly and essentially universally when dogs with lymphoma are treated with corticosteroids. We investigated naturally occurring mechanisms of prednisone resistance in seven dogs with naïve multicentric lymphoma, treated with oral prednisone; four dogs were administered concurrent cytotoxic chemotherapy. Expression of NR3C1α, ABCB1 (formerly MDR1), 11ß-HSD1, and 11ß-HSD2 mRNA was evaluated in neoplastic lymph nodes by real-time RT-PCR. Changes of expression levels at diagnosis and at time of clinical resistance to prednisone were compared longitudinally using a Wilcoxon signed-rank test. Clinical resistance to prednisone was observed after a median of 68 days (range: 7-348 days) after initiation of treatment. Relative to pretreatment samples, prednisone resistance was associated with decreased NR3C1α expression in biopsies of all dogs with high-grade lymphoma (six dogs, p=.031); one dog with indolent T-zone lymphoma had increased expression of NR3C1α. Resistance was not consistently associated with changes in ABCB1, 11ß-HSD1, or 11ß-HSD2 expression. Decreased expression of the glucocorticoid receptor (NR3C1α) may play a role in conferring resistance to prednisone in dogs with lymphoma. Results do not indicate a broad role for changes in expression of ABCB1, 11ß-HSD1, and 11ß-HSD2 in the emergence of prednisone resistance in lymphoma-bearing dogs.

Metronomic administration of lomustine following palliative radiation therapy for appendicular osteosarcoma in dogs.

The purpose of this study was to determine if metronomic administration of lomustine following palliative radiation therapy (RT) improved length of palliation and therefore survival in dogs with appendicular osteosarcoma compared to treatment with palliative radiation alone. A search of medical records identified dogs with appendicular osteosarcoma, treated with palliative RT (2 fractions of 8 Gray in a 24 hour time frame, day 0 and day 1; or day 0, 6 hours apart). Data collected included signalment, history, clinical signs, physical examination findings, clinicopathologic abnormalities, extent of disease, response, toxicity, other therapy, survival time, and whether dogs received metronomic lomustine (ML) or not. Of 86 patients, 43 received ML while 43 did not. Median survival time (MST) was not significantly different (P = 0.84), at 184 +/- 17 days for patients which received ML, and 154 +/- 20 days for those which did not. Metronomic lomustine administration was well-tolerated, but it did not improve survival in dogs with palliatively treated osteosarcoma.

Administration métronomique de lomustine après radiothérapie palliative pour le traitement des ostéosarcomes appendiculaires chez le chien. L'objectif de cette étude était de déterminer si l'administration métronomique de lomustine après radiothérapie palliative (RT) améliore la durée de palliation, et par conséquent la durée de vie, des chiens atteints d'ostéosarcome appendiculaire, en comparaison avec la radiothérapie seule. Les dossiers médicaux des chiens atteints d'ostéosarcome appendiculaire traités par radiothérapie palliative (2 fractions de 8 Gray dans un intervalle de 24 heures, jour 0 et jour 1; ou jour 0, à 6 heures d'intervalle) ont été identifiés et évalués. Les données collectées incluaient l'anamnèse, les commémoratifs, les anomalies de l'examen clinique et des analyses de laboratoires, les résultats du bilan d'extension, la réponse au(x) traitement(s), le développement de toxicités, d'éventuelles autres thérapies prodiguées, la durée de vie et si les chiens avaient été traités avec de la lomustine ou non. Sur 86 patients, 43 ont reçu de la lomustine tandis que 43 n'en ont pas reçu. La médiane de survie (MST) n'était pas significativement différente (P = 0.84), 184 +/− 17 jours pour les patients traités avec de la lomustine, et 154 +/− 20 jours pour ceux n'ayant pas reçu de lomustine. L'administration métronomique de lomustine était bien tolérée mais ne prodigua pas d'amélioration de la durée de vie lors de la prise en charge palliative des chiens atteints d'ostéosarcome.(Traduit par les auteurs).

Nonclinical Profile of BLZ-100, a Tumor-Targeting Fluorescent Imaging Agent.

BLZ-100 is a single intravenous use, fluorescent imaging agent that labels tumor tissue to enable more complete and precise surgical resection. It is composed of a chlorotoxin peptide covalently bound to the near-infrared fluorophore indocyanine green. BLZ-100 is in clinical development for intraoperative visualization of human tumors. The nonclinical safety and pharmacokinetic (PK) profile of BLZ-100 was evaluated in mice, rats, canines, and nonhuman primates (NHP). Single bolus intravenous administration of BLZ-100 was well tolerated, and no adverse changes were observed in cardiovascular safety pharmacology, PK, and toxicology studies in rats and NHP. The single-dose no-observed-adverse-effect-levels (NOAELs) were 7 mg (28 mg/kg) in rats and 60 mg (20 mg/kg) in NHP, corresponding to peak concentration values of 89 400 and 436 000 ng/mL and area-under-the-curve exposure values of 130 000 and 1 240 000 h·ng/mL, respectively. Based on a human imaging dose of 3 mg, dose safety margins are >100 for rat and monkey. BLZ-100 produced hypersensitivity reactions in canine imaging studies (lethargy, pruritus, swollen muzzle, etc). The severity of the reactions was not dose related. In a follow-up study in dogs, plasma histamine concentrations were increased 5 to 60 minutes after BLZ-100 injection; this coincided with signs of hypersensitivity, supporting the conclusion that the reactions were histamine based. Hypersensitivity reactions were not observed in other species or in BLZ-100 human clinical studies conducted to date. The combined imaging, safety pharmacology, PK, and toxicology studies contributed to an extensive initial nonclinical profile for BLZ-100, supporting first-in-human clinical trials.

TOLERABILITY AND TUMOR RESPONSE OF A NOVEL LOW-DOSE PALLIATIVE RADIATION THERAPY PROTOCOL IN DOGS WITH TRANSITIONAL CELL CARCINOMA OF THE BLADDER AND URETHRA.

Previously reported radiation protocols for transitional cell carcinoma of the canine lower urinary tract have been ineffective or associated with increased side effects. Objectives of this retrospective, cross-sectional study were to describe safety of and tumor responses for a novel palliative radiation protocol for transitional cell carcinoma in dogs. Included dogs had cytologically or histologically confirmed transitional cell carcinoma of the bladder or urethra, and were treated with 10 once-daily fractions (Monday-Friday) of 2.7 Gy. Thirteen dogs were sampled, with six treated using radiation as first-line (induction) therapy and seven treated using radiation as rescue therapy after failing previous chemotherapy. Within 6 weeks of radiation, 7.6% (1/13) dogs had a complete response, 53.8% (7/13) partial response, 38.5% (5/13) stable disease, and none had progressive disease. Three patients presenting with urethral obstruction had spontaneous micturition restored during the treatment protocol. A single patient with unilateral ureteral obstruction was patent at recheck examination. Median survival time from time of initial diagnosis was 179 days. Median survival time from start of radiation was 150 days. Acute radiation side effects occurred in 31% (4/13) patients and were classified as grade 1 or 2. No significant late side radiation side effects were reported. No variables examined were identified as prognostic factors. Findings indicated that the reported radiation protocol was safe in this sample of dogs with bladder and urethral transitional cell carcinoma. Future prospective studies are needed to determine utility of this treatment as a rescue therapy in patients with complete urinary tract obstruction.

Comparative whole transcriptome analysis of gene expression in three canine soft tissue sarcoma types.

Soft tissue sarcomas are pleiotropic tumors of mesenchymal cell origin. These tumors are rare in humans but common in veterinary practice, where they comprise up to 15% of canine skin and subcutaneous cancers. Because they present similar morphologies, primary sites, and growth characteristics, they are treated similarly, generally by surgical resection followed by radiation therapy. Previous studies have examined a variety of genetic changes as potential drivers of tumorigenesis and progression in soft tissue sarcomas as well as their use as markers for soft tissue sarcoma subtypes. However, few studies employing next generation sequencing approaches have been published. Here, we have examined gene expression patterns in canine soft tissue sarcomas using RNA-seq analysis of samples obtained from archived formalin-fixed and paraffin-embedded tumors. We provide a computational framework for using resulting data to categorize tumors, perform cross species comparisons and identify genetic changes associated with tumorigenesis. Functional overrepresentation analysis of differentially expressed genes further implicate both common and tumor-type specific transcription factors as potential mediators of tumorigenesis and aggression. Implications for tumor-type specific therapies are discussed. Our results illustrate the potential utility of this approach for the discovery of new therapeutic approaches to the management of canine soft tissue sarcomas and support the view that both common and tumor-type specific mechanisms drive the development of these tumors.

A prospective, multi-centre, Veterinary Radiation Therapy Oncology Group study reveals potential efficacy of toceranib phosphate (Palladia) as a primary or adjuvant agent in the treatment of canine nasal carcinoma.

Radiation is the standard of care for dogs with nasal tumours. The addition of another therapy that could improve outcome without increasing toxicity is attractive. Medical therapy that could offer better outcome than maximally tolerated dose chemotherapy when radiation therapy (RT) is not possible or is declined is also attractive. This article reports the findings from a prospective, multi-centre, non-randomized, Veterinary Radiation Therapy Oncology Group clinical trial designed to evaluate whether toceranib phosphate (toceranib) has primary activity and if the addition of toceranib to RT could positively impact outcome. Owner's discretion determined enrolment in toceranib alone or toceranib + RT arm. Historical controls for radiation alone were selected from patients treated with identical RT and imaging protocols. Responses were evaluated with pre-treatment and week-16 CT scans. RT total dose of 42 Gy was completed in 10 fractions. Sixty-three dogs enrolled from 10 study sites. Overall response rates (CR + PR) were significantly improved in the toceranib + RT (79.4%) and RT alone (68.9%) arms over toceranib alone (22%) (p = .011). Clinical benefit rates (CR + PR + SD) were significantly improved in the toceranib + RT arm over the RT alone arm at 97.3% and 79.2% respectively (p = .036). Treatment with toceranib alone, toceranib + RT and RT alone resulted in median survival times of 298, 615 and 368 days respectively, but were not statistically significantly different (p = .0502). Adverse events associated with toceranib administration did not potentiate the RT side effect profile. Toceranib appears to have primary activity against nasal carcinoma.

Adjuvant Sirolimus Does Not Improve Outcome in Pet Dogs Receiving Standard-of-Care Therapy for Appendicular Osteosarcoma: A Prospective, Randomized Trial of 324 Dogs.

PURPOSE: The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression. PATIENTS AND METHODS: A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (DFI), as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2-year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes. RESULTS: There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days [95% confidence interval (CI), 144-237] and 282 days (95% CI, 224-383) and for SOC + sirolimus dogs, it was 204 days (95% CI, 157-217) and 280 days (95% CI, 252-332), respectively. CONCLUSIONS: In a population of pet dogs nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.

Direct Interstitial Treatment of Solid Tumors Using an Injectable Yttrium-90-Polymer Composite.

Purpose: Yttrium-90 (90Y)-polymer composite (radiogel) may be administered directly into cancerous tissues to deliver highly localized beta radiation for therapy. In a dose-escalation study, the authors investigated the feasibility of treating feline and canine soft-tissue sarcomas as a model for nonresectable solid tumors in humans to gain clinical experience and to identify optimal methods for placing the composite uniformly within target tumor tissue. Materials and Methods: Five cats (Washington State University) and three dogs (University of Missouri) were selected for treatment from among veterinary clinic patients presenting with subcutaneous soft-tissue sarcomas. The therapeutic radiogel composite comprised two parts that were combined before therapy: (1) a calibrated activity of highly insoluble 90Y(YPO4) particles in a sterile, phosphate-buffered saline solution and (2) a resorbable hydrogel delivery vehicle containing a dissolved copolymer of poly-(DL-lactic acid-co-glycolic acid) and poly-(ethylene glycol). Sarcomas of anesthetized animals (five cats and three dogs) were injected with the 90Y-radiogel (10%-15% by tumor volume) using a parallel-needle grid pattern with ∼4-5-mm spacings with or without ultrasound guidance. After injection, the composite solution gelled within tumor interstitial spaces to solid phase upon reaching body temperatures to constrain the 90Y activity intratumorally. The animals were then imaged with computed tomography (CT) or positron emission tomography (PET)/CT and placed in radiation isolation for overnight monitoring and follow-up. Results: Gelation of the composite within tumor extracellular spaces confined the 90Y(YPO4) particles in place to deliver a planned radiation absorbed dose (100-320 Gy) to target tissue through complete decay. Response of the tumor tissue to 90Y-radiation therapy postexcision was evaluated by imaging, tumor resection, and histology. Correlation was observed on histopathology between tumor destruction and radiation dose. With uniform placement at high dose, the authors achieved complete remission or stable disease (at 1-2 months posttreatment). Conclusions: This study demonstrated successful injection of 90Y-polymer composite (radiogel) without discernable radiation dose to normal organs or other detrimental side effects. Animal patients recovered quickly from the injection procedure. The better therapeutic responses were observed at mean doses at or above 300 Gy.

An expedited palliative radiation protocol for lytic or proliferative lesions of appendicular bone in dogs.

Fifty-eight dogs with lytic or proliferative bone lesions were treated with a radiation protocol of two 8-Gy fractions over 2 consecutive days. The protocol was well tolerated, with no increase in early or late effects over previously published protocols. Forty-three (91%) of 47 dogs responded positively to radiation, with a median time of 2 days to onset of pain relief. Median duration of pain relief was 67 days (range 12 to 503 days; mean 99+/-16 days). Median survival time for all dogs was 136 days (mean 179+/-18 days). Distal radial location was a positive prognostic indicator for survival (P=0.005).

Granulocytopenia associated with thymoma in a domestic shorthaired cat.

A 5-year-old, spayed female cat was referred because of a mass in the cranial mediastinum noted on thoracic radiographs. A thymoma was diagnosed following ultrasound and biopsy of the mass. Treatment was initiated with coarse-fraction radiation therapy using external-beam therapy (four fractions of 5 Gy). The mass responded, but granulocytopenia developed. Bone marrow examination showed a myeloid to erythroid ratio of approximately 1:1, with a left shift within the myeloid line. These findings, as well as the lack of toxic changes within the peripheral blood neutrophils, suggested immune-mediated destruction of peripheral granulocytes. Immune suppression with prednisone and cyclosporine was instituted. After 7 weeks, the neutrophil count returned to normal. The tumor was removed, and cyclosporine was reduced and eventually discontinued 3 weeks postsurgery.

Reevaluation of the University of Wisconsin 2-year protocol for treating canine lymphosarcoma.

This retrospective study investigated a population of 96 dogs with newly diagnosed malignant lymphosarcoma that were treated with the commonly used University of Wisconsin-Madison (UW-M) chemotherapy protocol. Pretreatment characteristics were analyzed to determine prognostic factors. Dogs with higher World Health Organization (WHO) stages (including stage IV) and dogs with hypercalcemia were at significantly higher risk of relapse (P=0.018 and P=0.016, respectively). Dose reduction, treatment delays, and prior therapy with cortico-steroids were not associated with clinical outcome. First remission duration of 270 days was similar to historically reported data. Overall survival time of 218 days was much shorter than historical data.

Intranasal malignant melanoma in a dog.

A 10-year-old, female Newfoundland-cross dog was presented for evaluation of chronic intermittent unilateral epistaxis, nasal stertor, and sneezing. Nasal magnetic resonance imaging revealed a 3 x 5-cm mass in the left nasal cavity. Histopathological evaluation of nasal biopsies determined that the mass was a malignant melanoma. The mass was surgically resected and treated with bilateral opposed photon-beam radiation. This is the first report to describe the presentation, diagnosis, and treatment of an intranasal malignant melanoma in a dog.

Rostral maxillectomy and radiation therapy to manage squamous cell carcinoma in a ferret.

A 4-year-old, male, neutered ferret presented with squamous cell carcinoma of the right maxillary region associated with the tissues surrounding the upper canine tooth. A rostral maxillectomy was performed to excise the mass. Histopathologic examination showed questionable margins of tumor removal. Approximately 2 months after surgery, the ferret received a course of radiation therapy and is currently being monitored for tumor regrowth.

Preclinical Validation of the Utility of BLZ-100 in Providing Fluorescence Contrast for Imaging Spontaneous Solid Tumors.

There is a need in surgical oncology for contrast agents that can enable real-time intraoperative visualization of solid tumors that can enable complete resections while sparing normal surrounding tissues. The Tumor Paint agent BLZ-100 is a peptide-fluorophore conjugate that can specifically bind solid tumors and fluoresce in the near-infrared range, minimizing light scatter and signal attenuation. In this study, we provide a preclinical proof of concept for use of this imaging contrast agent as administered before surgery to dogs with a variety of naturally occurring spontaneous tumors. Imaging was performed on excised tissues as well as intraoperatively in a subset of cases. Actionable contrast was achieved between tumor tissue and surrounding normal tissues in adenocarcinomas, squamous cell carcinomas, mast cell tumors, and soft tissue sarcomas. Subcutaneous soft tissue sarcomas were labeled with the highest fluorescence intensity and greatest tumor-to-background signal ratio. Our results establish a foundation that rationalizes clinical studies in humans with soft tissue sarcoma, an indication with a notably high unmet need.

Ultrasound guided, pre-radiation oxygen measurements using polarographic oxygen needle electrodes in spontaneous canine soft tissue sarcomas.

BACKGROUND: Poor tumour oxygenation is associated with radiation resistance. The recording of pre-treatment oxygenation status has been shown to be of prognostic relevance. MATERIAL AND METHODS: Eleven dogs with spontaneously arising soft tissue sarcomas were included in this study. Oxygen partial pressure measurements (pO2) were performed with the Eppendorf method. RESULTS: The mean of median pO2 was 9.6 mmHg (range: 0.1-30 mmHg). Four of the nine dogs included in the statistical analysis showed a median pO2 < or = 2.5 mmHg. The natural logarithm of the hypoxic subvolume correlated with the hypoxic fraction < or = 2.5 mmHg (p = 0.0712) and < or 5 mmHg (p = 0.0988). Mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) correlated significantly to several oxygen parameters. CONCLUSION: Hypoxia exists in spontaneous canine soft tissue sarcomas and the dog can be used as a reliable model for repeated oxygenation measurements. Ultrasonography assures reliability of needle placement.

Evaluation of vincristine-associated myelosuppression in Border Collies.

OBJECTIVE: To determine whether Border Collies (ATP binding cassette subfamily B1 gene [ABCB1] wildtype) were more likely than other breeds to develop vincristine-associated myelosuppression (VAM) and, if so, whether this was caused by a mutation in ABCB1 distinct from ABCB1-1Δ. ANIMALS: Phase 1 comprised 36 dogs with the ABCB1 wildtype, including 26 dogs with lymphoma (5 Border Collies and 21 dogs representing 13 other breeds) treated with vincristine in a previous study; phase 2 comprised 10 additional Border Collies, including 3 that developed VAM and 7 with an unknown phenotype. PROCEDURES: For phase 1, the prevalence of VAM in ABCB1-wildtype Border Collies was compared with that for ABCB1-wildtype dogs of other breeds with data from a previous study. For phase 2, additional Border Collies were included. Hematologic adverse reactions were graded with Veterinary Co-operative Oncology Group criteria. Genomic DNA was used to amplify and sequence all 27 exons of the canine ABCB1. Sequences from affected dogs were compared with those of unaffected dogs and dogs of unknown phenotype. RESULTS: 3 of 5 Border Collies with the ABCB1 wildtype developed VAM; this was significantly higher than the proportion of other dogs that developed VAM (0/21). A causative mutation for VAM in Border Collies was not identified, although 8 single nucleotide polymorphisms in ABCB1 were detected. CONCLUSIONS AND CLINICAL RELEVANCE: Breed-associated sensitivity to vincristine unrelated to ABCB1 was detected in Border Collies. Veterinarians should be aware of this breed predisposition to VAM. Causes for this apparent breed-associated sensitivity should be explored.

Correlation of ultrasound findings, liver and spleen cytology, and prognosis in the clinical staging of high metastatic risk canine mast cell tumors.

Cytologic sampling of the ultrasonographically normal spleen and liver is not implemented routinely in the clinical staging of canine cutaneous mast cell tumors and normal ultrasound findings are often accepted as sufficient evidence for ruling out splenic or liver metastasis. Our objective was to define the specificity and sensitivity of ultrasound findings for diagnosis of mast cell infiltration when verified with cytologic evaluation, and to define the prognostic role of cytologic evaluation of liver and splenic aspirates. Dogs with a diagnosis of clinically aggressive grade II, or grade III mast cell tumor treated with a combination vinblastine/CCNU chemotherapy protocol, were selected retrospectively based on availability of cytologic evaluation of spleen plus or minus liver for staging. Out of 19 dogs, 10 dogs had a grade II tumor and nine a grade III tumor. Seven dogs had mast cell infiltration of the spleen, liver, or both. The sensitivity of ultrasound for detecting mast cell infiltration was 43% for the spleen and 0% for the liver. Dogs with positive cytologic evidence of mast cell infiltration to spleen, liver, or both had significantly shorter survival (100 vs. 291 days) than dogs without evidence of mast cell infiltration (P<0.0001). Routine splenic aspiration should be performed regardless of ultrasonographic appearance in dogs with a clinically aggressive mast cell tumor.

Radiation therapy in horses.

Although the diagnosis of cancer is relatively uncommon in horses, tumors do occur in this species. Surgery, radiation, and chemotherapy are traditional cancer treatments in all species. In equine patients, surgery has often been the only treatment offered; however, not all tumors can be controlled with surgery alone. In small animal oncology, newer and better therapies are in demand and available. Radiation therapy is often used to control or palliate tumors locally, especially to satisfy clients who demand sophisticated treatments. The large size of equine patients can make radiation therapy difficult, but it is a valuable tool for treating cancer and should not be overlooked when treating horses.