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Fluorescence imaging is increasingly being implemented in surgery. One of the drawbacks of its application is the need to switch back-and-forth between fluorescence- and white-light-imaging settings and not being able to dissect safely under fluorescence guidance. The aim of this study was to engineer 'click-on' fluorescence detectors that transform standard robotic instruments into molecular sensing devices that enable the surgeon to detect near-infrared (NIR) fluorescence in a white-light setting. This NIR-fluorescence detector setup was engineered to be press-fitted onto standard forceps instruments of the da Vinci robot. Following system characterization in a phantom setting (i.e., spectral properties, sensitivity and tissue signal attenuation), the performance with regard to different clinical indocyanine green (ICG) indications (e.g., angiography and lymphatic mapping) was determined via robotic surgery in pigs. To evaluate in-human applicability, the setup was also used for ICG-containing lymph node specimens from robotic prostate cancer surgery. The resulting Click-On device allowed for NIR ICG signal identification down to a concentration of 4.77 × 10 -6  mg/ml. The fully assembled system could be introduced through the trocar and grasping, and movement abilities of the instrument were preserved. During surgery, the system allowed for the identification of blood vessels and assessment of vascularization (i.e., bowel, bladder and kidney), as well as localization of pelvic lymph nodes. During human specimen evaluation, it was able to distinguish sentinel from non-sentinel lymph nodes. With this introduction of a NIR-fluorescence Click-On sensing detector, a next step is made towards using surgical instruments in the characterization of molecular tissue aspects., (© 2022. The Author(s).)

Purpose: Sentinel lymph node biopsy is a routine procedure for nodal staging in penile cancer. Most commonly, this procedure is guided by radioactive tracers, providing various forms of preoperative and intraoperative guidance. This is further extended with fluorescence imaging using hybrid radioactive-fluorescence tracers. Alternatively, a magnetic-based approach has become available using superparamagnetic iron-oxide nanoparticles (SPIONs). This study investigates a novel freehand magnetic particle imaging and navigation modality (fhMPI) for intraoperative localization, along with a hybrid approach, combining magnetic and fluorescence guidance., Materials and Methods: The fhMPI set-up was built with a surgical navigation device, optical tracking system and magnetometer probe. A dedicated reconstruction software based on a look-up-table method was used to reconstruct a superficial 3D volume of the SPION distribution in tissue. For fluorescence guidance, indocyanine green (ICG) was added to the SPIONs. The fhMPI modality was characterized in phantoms, ex vivo human skin and in vivo porcine surgery., Results: Phantom and human skin explants illustrated that the current fhMPI modality had a sensitivity of 2.2 × 10 -2  mg/mL SPIONs, a resolving power of at least 7 mm and a depth penetration up to 1.5 cm. Evaluation during porcine surgery showed that fhMPI allowed for an augmented reality image overlay of the tracer distribution in tissue, as well as 3D virtual navigation. Besides, using the hybrid approach, fluorescence imaging provided a visual confirmation of localized nodes., Conclusion: fhMPI is feasible in vivo, providing 3D imaging and navigation for magnetic nanoparticles in the operating room, expanding the guidance possibilities during magnetic sentinel lymph node procedures. Furthermore, the integration of ICG provides the ability to visually refine and confirm correct localization. Further clinical evaluation should verify these findings in human patients as well., (© 2021. The Author(s).)

The field of fluorescence-guided surgery builds on colored fluorescent tracers that have become available for different clinical applications. Combined use of complementary fluorescent emissions can allow visualization of different anatomical structures (e.g. tumor, lymphatics and nerves) in the same patient. With the aim to assess the requirements for multi-color fluorescence guidance under in vivo conditions, we thoroughly characterized two FDA-approved laparoscopic Firefly camera systems available on the da Vinci Si or da Vinci Xi surgical robot. In this process, we studied the cameras' performance with respect to the photophysical properties of the FDA-approved dyes Fluorescein and ICG. Our findings indicate that multi-wavelength fluorescence imaging of Fluorescein and ICG is possible using clinical-grade fluorescence laparoscopes, but critical factors for success include the photophysical dye properties, imaging system performance and the amount of accumulated dye. When comparing the camera performance, the Xi system provided more effective excitation (adaptions in the light source) and higher detection sensitivity (chip-on-a-tip and/or enhanced image processing) for both Fluorescein and ICG. Both systems can readily be used for multi-wavelength fluorescence imaging of Fluorescein and ICG under clinically relevant conditions. With that, another step has been made towards the routine implementation of multi-wavelength image-guided surgery concepts., (© 2020. The Author(s).)

Robot-assisted radical prostatectomy (RARP) is performed in patients with prostate cancer. Unfortunately, 10-46% of patients may still suffer from limited erectile function (EF) after RARP. This study aimed to develop a prediction model based on the extent of fascia preservation (FP) and postoperative EF after RARP. A previously developed FP score quantizing the extent and regions of nerve-preservation was determined in a cohort of 1241 patients who underwent RARP. The predictive value of the FP score for post-prostatectomy EF (following the international index erectile function (IIEF) score, EF domain) was analyzed. To increase the predictive value of the scoring system, the FP regions were related to postoperative EF, nerve distribution and co-morbidity factors. Finally, a prediction model for EF was developed based on the studied cohort. When corrected for the preoperative IIEF-EF, the FP score was shown to be a significant denominator for IIEF (p = 2.5 × 10 - 15 ) with an R 2 of 35%. Variable selection performed using the Akaike information criterion led to a final prediction model for postoperative IIEF after nerve-preservation based on the FP score. Furthermore, patient's age, preoperative IIEF score, CCIS and use of clips for nerve sparing were significantly associated with postoperative IIEF-EF. More anterior fascia preservation was correlated with better EF outcome and age was a strong independent predictor of EF outcome. In older men, the relative benefit of more extensive fascia preservation was at least similar to younger men, despite a lower baseline IIEF-EF score. Quantitative nerve-sparing FP scoring could be related to the postoperative IIEF-EF and integrated into a multivariate prediction model, which includes with age, use of surgical clips, the Charlson Comorbidity Index Score (CCIS), and preoperative IIEF-EF. When further validated the prediction model could provide patients and care-givers a qualitative estimation of EF outcome after RARP.

Purpose: This study was designed to examine the feasibility of combining lymphoscintigraphy and intraoperative sentinel node identification in patients with head and neck melanoma by using a hybrid protein colloid that is both radioactive and fluorescent., Methods: Eleven patients scheduled for sentinel node biopsy in the head and neck region were studied. Approximately 5 h before surgery, the hybrid nanocolloid labeled with indocyanine green (ICG) and technetium-99m ((99m)Tc) was injected intradermally in four deposits around the scar of the primary melanoma excision. Subsequent lymphoscintigraphy and single photon emission computed tomography with computed tomography (SPECT/CT) were performed to identify the sentinel nodes preoperatively. In the operating room, patent blue dye was injected in 7 of the 11 patients. Intraoperatively, sentinel nodes were acoustically localized with a gamma ray detection probe and visualized by using patent blue dye and/or fluorescence-based tracing with a dedicated near-infrared light camera. A portable gamma camera was used before and after sentinel node excision to confirm excision of all sentinel nodes., Results: A total of 27 sentinel nodes were preoperatively identified on the lymphoscintigraphy and SPECT/CT images. All sentinel nodes could be localized intraoperatively. In the seven patients in whom blue dye was used, 43% of the sentinel nodes stained blue, whereas all were fluorescent. The portable gamma camera identified additional sentinel nodes in two patients. Ex vivo, all radioactive lymph nodes were fluorescent and vice versa, indicating the stability of the hybrid tracer., Conclusions: ICG-(99m)Tc-nanocolloid allows for preoperative sentinel node visualization and concomitant intraoperative radio- and fluorescence guidance to the same sentinel nodes in head and neck melanoma patients.

Purpose: Erlotinib (Tarceva®, OSI-774) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. As high-grade gliomas frequently show amplification, overexpression and/or mutation of EGFR, this drug has been tested in several clinical trials with glioblastoma patients, but unfortunately, with little success. As erlotinib is a known substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) we have investigated the effect of these ABC-transporters on the brain penetration of erlotinib., Study Design: Erlotinib (50 mg/kg) was given by i.p. administration to wild-type (WT), Mdr1ab(-/-) (single P-gp knockout), Bcrp1(-/-) (single Bcrp1 knockout) and Mdr1ab(-/-)Bcrp1(-/-) (compound P-gp and Bcrp1 knockout) mice. Drug levels in plasma and tissues were determined by reversed-phase high-performance liquid chromatography., Results: Relative to Mdr1ab(-/-)Bcrp1(-/-) mice that are deficient for both drug transporters, the area under the concentration time curve in brain tissue (AUC)(brain) of erlotinib decreased significantly by 1.6-fold in Mdr1ab(-/-) mice where Bcrp1 is present (49.6 ± 3.95 versus 31.1 ± 1.7, μg/g*h; P < 0.01). In Bcrp1(-/-) mice, were P-gp is present, a more pronounced 3.8-fold decrease to 13.0 ± 0.70, μg/g*h (P < 0.01) was observed, which is close to the 4.5-fold decrease in the AUC(brain) of erlotinib found in WT mice where both drug transporters are present (11.0 ± 1.35, P < 0.01). The plasma clearance of erlotinib was similar in mice deficient for P-gp and/or Bcrp1 compared with wild-type mice. In all other tissues the differences between the genotypes were negligible., Conclusions: Both P-gp and Bcrp1 reduce the brain penetration of erlotinib. Although P-gp appears to be the most effective factor limiting the brain penetration of erlotinib, the highest brain accumulation was observed when Bcrp1 was also absent. Strategies to inhibit P-gp/BCRP in patients to improve delivery of (novel molecular-targeted) substrate agents, such as erlotinib, to the brain may be required for treatment of intracranial malignancies.

The anticancer drug paclitaxel is formulated for i.v. administration in a mixture of Cremophor EL and ethanol.Its oral bioavailability is very low due to the action of P-glycoproteinin the gut wall and CYP450 in gut wall and liver.However, proof-of-concept studies using the i.v. formulation diluted in drinking water have demonstrated the feasibility of the oral route as an alternative when given in combination with inhibitors of P-glycoprotein and CYP450. Because of the unacceptable pharmaceutical properties of the drinking solution, a better formulation for oral application is needed.We have evaluated the suitability of various self-micro emulsifying oily formulations (SMEOF's) of paclitaxel for oral application using wild-type and P-glycoprotein knockout mice and cyclosporin A (CsA) as P-glycoprotein and CYP450 inhibitor. The oral bioavailability of paclitaxel in all SMEOF's without concomitant CsA was low in wild-type mice, showing that this vehicle does not enhance intestinal uptake by itself.Paclitaxel (10 mg/kg) in SMEOF#3 given with CsA resulted in plasma levels that were comparable to the Cremophor ELethanol containing drinking solution plus CsA. Whereas the AUC increased linearly with the oral paclitaxel dose in P-glycoprotein knockout mice, it increased less than proportional in wild-type mice given with CsA. In both strains more unchanged paclitaxel was recovered in the feces at higher doses. This observation most likely reflects more profound precipitation of paclitaxel within the gastro-intestinal tract at higher doses. The resulting absolute reduction in absorption of paclitaxel from the gut was possibly concealed by partial saturation of first-pass metabolism when P-glycoprotein was absent. In conclusion, SMEOF's maybe a useful vehicle for oral delivery of paclitaxel in combination with CsA, although the physical stability within the gastro-intestinal tract remains a critical issue, especially when applied at higher dose levels.

Trabectedin is a novel anticancer drug active against soft tissue sarcomas. Trabectedin is a substrate for P-glycoprotein (P-gp), which is encoded by mdr1a/1b in rodents. Plasma and tissue distribution, and excretion of [(14)C]-trabectedin were evaluated in wild-type and mdr1a/1b(-/-) mice. In parallel, we investigated the toxicity profile of trabectedin by serial measurements of blood liver enzymes and general pathology. [(14)C]-trabectedin was extensively distributed into tissues, and rapidly converted into a range of unknown metabolic products. The excretion of radioactivity was similar in both genotypes. The plasma clearance of unchanged trabectedin was not reduced when P-gp was absent, but organs under wild type circumstances protected by P-gp showed increased trabectedin concentrations in mdr1a/1b(-/-) mice. Although hepatic trabectedin concentrations were not increased when P-gp was absent, mdr1a/1b(-/-) mice experienced more severe liver toxicity. P-gp plays a role in the in vivo disposition and toxicology of trabectedin.

Imatinib is transported by P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), however, the exact impact of these transporters on absorption, distribution, metabolism and excretion (ADME) of imatinib is not fully understood due to incomplete data. We have performed a comprehensive ADME study of imatinib given as single agent or in combination with the well known BCRP/P-gp inhibitors, elacridar and pantoprazole, in wild-type and P-gp and/or BCRP knockout mice. The absence of P-gp and BCRP together resulted in a significantly higher area under the plasma concentration-time curve (AUC) after i.v. administration, whereas the AUC after oral dosing was unaltered. Both elacridar and pantoprazole significantly increased the AUC of orally administered imatinib in wild-type but also in P-gp/BCRP knockout mice. This lower clearance was not due to a (further) reduction in biliary excretion. Fecal excretion was significantly reduced in P-gp and P-gp/BCRP knockout but not in BCRP knockout mice, whereas the brain penetration was significantly higher in P-gp/BCRP knockout mice compared to single P-gp or BCRP knockout or wild-type mice. In conclusion, P-gp and BCRP have only a modest effect on the ADME of imatinib in comparison to metabolic elimination. P-gp is the most prevalent factor for systemic clearance and limiting the brain penetration. The considerable drug-drug interaction observed with elacridar or pantoprazole is only partly mediated by inhibition of P-gp and BCRP and far more by the inhibition of other elimination pathways.

Trabectedin (ET-743, Yondelis) is a novel anticancer drug currently undergoing phase II and III investigations. There are various and conflicting reports whether trabectedin is a substrate for P-glycoprotein (P-gp), an important factor in drug disposition and multi-drug resistance (MDR). We have now unambiguously shown that trabectedin is a P-gp substrate by investigating vectorial transport over monolayers of LLC-PK1 pig kidney and Madine-Darby Canine kidney (MDCK) cells and the mdr1a and/or MDR1 transfected subclones. We further characterized the cytotoxic effects and cellular accumulation of trabectedin in these cell lines as well as in a panel of other cell lines with high or moderate expression levels of P-gp. Trabectedin displayed the typical MDR phenotype only in highly P-gp expressing cell lines, but not in cell lines with expression levels more closely conforming to clinical samples, suggesting that P-gp will not confer resistance to trabectedin in cancer patients.