•A sensitive HPLC-MS/MS method was developed for the quantification of miltefosine in human skin tissue.•An enzymatic digestion method homogenized human skin tissues before protein precipitation and ...phenyl-SPE sample preparation.•Miltefosine was stable under homogenization conditions.•Human skin tissue homogenates were stable for a minimum of 10 days stored at − 20 °C.•There was no significant matrix effect from the human skin tissue matrix on the performance of the assay.
Miltefosine is the only oral drug approved for the treatment of various clinical presentations of the neglected parasitic disease leishmaniasis. In cutaneous leishmaniasis and post-kala-azar dermal leishmaniasis, Leishmania parasites reside and multiply in the dermis of the skin. As miltefosine is orally administered and this drug is currently studied for the treatment of these skin-related types of leishmaniasis, there is an urgent need for an accurate assay to determine actual miltefosine levels in human skin tissue to further optimize treatment regimens through target-site pharmacokinetic studies. We describe here the development and validation of a sensitive method to quantify miltefosine in 4-mm human skin biopsies utilizing high-performance liquid chromatography coupled to tandem mass spectrometry. After the skin tissues were homogenized overnight by enzymatic digestion using collagenase A, the skin homogenates were further processed by protein precipitation and phenyl-bonded solid phase extraction. Final extracts were injected onto a Gemini C18 column using alkaline eluent for separation and elution. Detection was performed by positive ion electrospray ionization followed by a quadrupole – linear ion trap mass spectrometer, using deuterated miltefosine as an internal standard. The method was validated over a linear calibration range of 4–1000 ng/mL (r2 ≥ 0.9996) using miltefosine spiked digestion solution for calibration and quality control samples. Validation parameters were all within internationally accepted criteria, including intra- and inter-assay accuracies and precisions within± 15% and ≤ 15% (within± 20% and ≤ 20% at the lower limit of quantitation). There was no significant matrix effect of the human skin tissue matrix and the recovery for miltefosine, and internal standard were comparable. Miltefosine in human skin tissue homogenates was stable during the homogenization incubation (37 °C,± 16 h) and after a minimum of 10 days of storage at − 20 °C after the homogenization process. With our assay we could successfully detect miltefosine in skin biopsies from patients with post-kala azar dermal leishmaniasis who were treated with this drug in Bangladesh.
Mutations in KRAS result in a constitutively activated MAPK pathway. In KRAS-mutant tumours existing treatment options, e.g. MEK inhibition, have limited efficacy due to resistance through feedback ...activation of epidermal growth factor receptors (HER).
In this Phase 1 study, the pan-HER inhibitor dacomitinib was combined with the MEK1/2 inhibitor PD-0325901 in patients with KRAS-mutant colorectal, pancreatic and non-small-cell lung cancer (NSCLC). Patients received escalating oral doses of once daily dacomitinib and twice daily PD-0325901 to determine the recommended Phase 2 dose (RP2D). (Clinicaltrials.gov: NCT02039336).
Eight out of 41 evaluable patients (27 colorectal cancer, 11 NSCLC and 3 pancreatic cancer) among 8 dose levels experienced dose-limiting toxicities. The RP2D with continuous dacomitinib dosing was 15 mg of dacomitinib plus 6 mg of PD-0325901 (21 days on/7 days off), but major toxicity, including rash (85%), diarrhoea (88%) and nausea (63%), precluded long-term treatment. Therefore, other intermittent schedules were explored, which only slightly improved toxicity. Tumour regression was seen in eight patients with the longest treatment duration (median 102 days) in NSCLC.
Although preliminary signs of antitumour activity in NSCLC were seen, we do not recommend further exploration of this combination in KRAS-mutant patients due to its negative safety profile.
Purpose
KRAS
oncogene mutations cause sustained signaling through the MAPK pathway. Concurrent inhibition of MEK, EGFR, and HER2 resulted in complete inhibition of tumor growth in
KRAS
-mutant (
KRAS
...m) and
PIK3CA
wild-type tumors, in vitro and in vivo
.
In this phase I study, patients with advanced
KRAS
m and
PIK3CA
wild-type colorectal cancer (CRC), non-small cell lung cancer (NSCLC), and pancreatic cancer, were treated with combined lapatinib and trametinib to assess the recommended phase 2 regimen (RP2R).
Methods
Patients received escalating doses of continuous or intermittent once daily (QD) orally administered lapatinib and trametinib, starting at 750 mg and 1 mg continuously, respectively.
Results
Thirty-four patients (16 CRC, 15 NSCLC, three pancreatic cancers) were enrolled across six dose levels and eight patients experienced dose-limiting toxicities, including grade 3 diarrhea (
n
= 2), rash (
n
= 2), nausea (
n
= 1), multiple grade 2 toxicities (
n
= 1), and aspartate aminotransferase elevation (
n
= 1), resulting in the inability to receive 75% of planned doses (
n
= 2) or treatment delay (
n
= 2). The RP2R with continuous dosing was 750 mg lapatinib QD plus 1 mg trametinib QD and with intermittent dosing 750 mg lapatinib QD and trametinib 1.5 mg QD 5 days on/2 days off. Regression of target lesions was seen in 6 of the 24 patients evaluable for response, with one confirmed partial response in NSCLC. Pharmacokinetic results were as expected.
Conclusion
Lapatinib and trametinib could be combined in an intermittent dosing schedule in patients with manageable toxicity. Preliminary signs of anti-tumor activity in NSCLC have been observed and pharmacodynamic target engagement was demonstrated.
Most patients with cancer treated with everolimus experience stomatitis, which seriously affects the quality of life. The salivary concentrations of everolimus may predict the incidence and severity ...of stomatitis. The authors aimed to examine whether it was feasible to quantify the everolimus concentration in saliva and subsequently use it to predict stomatitis.
Saliva and whole blood samples were taken from patients with cancer, who were treated with everolimus in the dosage of either 10 mg once a day or 5 mg twice a day. Everolimus concentrations in saliva samples were measured by liquid chromatography-tandem mass spectrometry. A published population pharmacokinetic model was extended with the everolimus concentration in saliva to assess any association between everolimus in the blood and saliva. Subsequently, the association between the occurrence of stomatitis and the everolimus concentration in saliva was studied.
Eleven patients were included in this study; saliva samples were available from 10 patients, including 3 patients with low-grade stomatitis. Everolimus concentrations were more than 100-fold lower in saliva than in whole blood (accumulation ratio 0.00801 and relative standard error 32.5%). Interindividual variability (67.7%) and residual unexplained variability (84.0%) were high. The salivary concentration of everolimus tended to be higher in patients with stomatitis, 1 hour postdose ( P = 0.14).
Quantification of the everolimus concentration in saliva was feasible and revealed a nonsignificant correlation between everolimus concentration in the saliva and the occurrence of stomatitis. If future research proves this relationship to be significant, the everolimus concentration in the saliva may be used as an early predictor of stomatitis without invasive sampling. Thereby, in patients with high salivary everolimus concentrations, precautions can be taken to decrease the incidence and severity of stomatitis.
There is a lack of understanding whether plasma levels of anticancer drugs (such as pazopanib) correlate with intra-tumoral levels and whether the plasma compartment is the best surrogate for ...pharmacokinetic and pharmacodynamic evaluation. Therefore, we aimed to quantify pazopanib concentrations in tumor tissue, to assess the correlation between tumor concentrations and plasma concentrations and between tumor concentrations and efficacy. In this clinical trial, non-metastatic STS patients were treated with neo-adjuvant concurrent radiotherapy and pazopanib. Plasma samples and tumor biopsies were collected, and pazopanib concentrations were measured using liquid chromatography-tandem mass spectrometry. Twenty-four evaluable patients were included. The median pazopanib tumor concentration was 19.2 µg/g (range 0.149–200 µg/g). A modest correlation was found between tumor concentrations and plasma levels of pazopanib (ρ = 0.41, p = 0.049). No correlation was found between tumor concentrations and percentage of viable tumor cells (p > 0.05); however, a trend towards less viable tumor cells in patients with high pazopanib concentrations in tumor tissue was observed in a categorical analysis. Possible explanations for the lack of correlation might be heterogeneity of the tumors and timing of the biopsy procedure.
Background
ModraDoc006 is an oral formulation of docetaxel, which is co‐administered with the cytochrome P450 3A4 and P‐glycoprotein inhibitor ritonavir (r): ModraDoc006/r. Weekly treatment with ...ModraDoc006/r had been evaluated in phase I trials in patients with different types of advanced solid tumors, but up to this point in time not in patients with metastatic castration‐resistant prostate cancer (mCRPC).
Aim
We assessed safety and pharmacokinetics (PK) of ModraDoc006/r to establish the recommended phase 2 dose (RP2D) in patients with mCRPC.
Methods
mCRPC patients, treatment naïve or following abiraterone or enzalutamide treatment, were included. Dose‐escalation of ModraDoc006/r was based on safety and docetaxel PK. Antitumor activity was assessed by serum prostate‐specific antigen (PSA) and radiological evaluation.
Results
Cohort 1 (n = 5) received once weekly ModraDoc006 30 mg with ritonavir 100 mg in the morning, and ModraDoc006 20 mg with ritonavir 100 mg in the evening (30‐20/100‐100). The mean docetaxel area under the plasma concentration‐time curve (mAUC0‐inf) was 461 ng/mL × h with 1 dose limiting toxicity (DLT); grade 3 alanine transferase increase. In cohort 2 (n = 6, ModraDoc006/r 30‐20/200‐200), the mAUC0‐inf was 1687 ng/mL × h with 2 DLTs; grade 3 diarrhea and mucositis. In cohort 3A (n = 6, ModraDoc006/r 30‐20/200‐100), the mAUC0‐inf was 1517 ng/mL × h with 1 DLT; grade 3 diarrhea. In cohort 3B (n = 3, ModraDoc006/r 20‐20/200‐100), the mAUC0‐inf was 558 ng/mL × h without DLTs. The mAUC0‐inf exceeded estimated exposures of intravenous docetaxel in cohort 2 and 3A, was lower in cohort 1 and was in range in cohort 3B. PSA decreases of >50% occurred in 6/10 evaluable patients throughout the various cohorts. In five radiological evaluable patients, two confirmed partial responses were observed.
Conclusion
The RP2D was established at weekly ModraDoc006/r 30‐20/200‐100. Observed PSA and radiological responses suggest promising clinical activity. These results have led to an ongoing randomized Phase 2b study, comparing weekly ModraDoc006/r with 3‐weekly IV docetaxel in patients with mCRPC.
•An accurate and precise bioanalytical quantification ofparomomycin in human skin tissue was achieved.•Enzymatical digestion of human skin tissue was performed before sample preparation.•Validation ...of the bioanalytical method was achieved following the EMA guidelines.•Post-kala-azar dermal leishmaniasis clinical skin biopsies were be quantified using this method.
Bioanalytical assay development and validation procedures were performed to quantify antiprotozoal drug paromomycin in human skin tissue by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Paromomycin, an aminoglycoside drug, is administered intra-muscularly and used in the treatment of multiple clinical presentations of the neglected tropical disease leishmaniasis. It is currently studied in the treatment of post-kala-azar dermal leishmaniasis, a disease where the Leishmania parasites divide and reside in the skin. We present a target-site bioanalytical method to accurately quantify paromomycin in human skin tissue, with the clinical purpose of quantifying paromomycin in skin biopsies from post-kala-azar dermal leishmaniasis patients originating from Sudan. Enzymatic digestion using collagenase A incubated at 37 °C overnight was employed as homogenization method to produce skin tissue homogenates. Further sample preparation was performed by protein precipitation using trichloroacetic acid and a dilution step. Final extracts were injected onto a C18 analytical column and isocratic heptafluorobutyric acid ion-pair separation and elution were employed. The chromatography system was coupled to a triple quadrupole mass spectrometer for detection. The method was validated in digestion solution over a linear range from 5 to 1000 ng/mL (r2 ≥ 0.9967) with the assay performance of accuracy and precision within acceptable criteria values as stated by the EMA guidelines. Furthermore, matrix effects were observed in human skin tissue and were corrected by the multiple deuterated paromomycin internal standard. No substantial IS-normalized matrix effect was detected along with relatively high sample preparation recovery. Consequently, digestion solution matrix serving as the preparation of calibration standards can be used as surrogate matrix for human skin tissue, which is convenient given the limited availability of control matrix. Finally, paromomycin was accurately quantified in skin of post-kala-azar dermal leishmaniasis patients originating from clinical trials in Sudan.
•An accurate and precise bioanalytical quantification of amphotericin B in human skin tissue was achieved.•Skin tissue sample preparation combining enzymatic digestion using collagenase A and simple ...protein precipitation was developed.•Human skin tissue homogenates were stable for a minimum of 3 days stored at −70 °C.•There was no significant IS-normalized matrix effect from skin tissue matrix on the performance of the assay.•Clinical skin biopsies could all be quantified using this method.
Amphotericin B is an antifungal and antiparasitic drug used in first-line treatment of the parasitic neglected tropical disease leishmaniasis. Liposomal amphotericin B is currently studied for the treatment of cutaneous and post-kala-azar dermal leishmaniasis, where the dermis of the skin is infected with Leishmania parasites. For the optimization of known treatment regimens, accurate target-site concentrations of the drug are required. To date, no assay was available to assess human skin concentrations of amphotericin B. We here present a bioanalytical assay for the quantification of amphotericin B in 4-mm human skin biopsies. Human skin biopsies were homogenized by overnight digestion using collagenase A and were processed afterwards by simple protein precipitation using methanol. Separation and detection were achieved using a Gemini C18 column with slightly acidic chromatographic conditions and a quadrupole – linear ion trap mass spectrometer, respectively. The method was validated in digestion solution over a range of 10–2,000 ng/mL using natamycin as internal standard, with a correlation coefficient (r2) of at least 0.9974. The assay performance, accuracy and precision, were acceptable over the validated range, using international (EMA and FDA) acceptance criteria. In the skin tissue extracts, amphotericin B ion enhancement was observed, however, the internal standard (IS) corrected for this effect hence calibration standards in digestion solvent could be used as a surrogate matrix for the quantification in skin tissue. Sample preparation recoveries were low (around 27%) because of degradation of amphotericin B during digestion and sample preparation processes, albeit highly reproducible, without compromising the accuracy and precision of the method. Using this assay, amphotericin B could be detected and quantified in skin biopsies originating from treated Indian post-kala-azar dermal leishmaniasis patients.
Lessons Learned
Afatinib and selumetinib can be combined in continuous and intermittent dosing schedules, albeit at lower doses than approved for monotherapy.
Maximum tolerated dose for continuous ...and intermittent schedules is afatinib 20 mg once daily and selumetinib 25 mg b.i.d.
Because the anticancer activity was limited, further development of this combination is not recommended until better biomarkers for response and resistance are defined.
Background
Antitumor effects of MEK inhibitors are limited in KRAS‐mutated tumors because of feedback activation of upstream epidermal growth factor receptors, which reactivates the MAPK and the phosphoinositide 3‐kinase–AKT pathway. Therefore, this phase I trial was initiated with the pan‐HER inhibitor afatinib plus the MEK inhibitor selumetinib in patients with KRAS mutant, PIK3CA wild‐type tumors.
Methods
Afatinib and selumetinib were administered according to a 3+3 design in continuous and intermittent schedules. The primary objective was safety, and the secondary objective was clinical efficacy.
Results
Twenty‐six patients were enrolled with colorectal cancer (n = 19), non‐small cell lung cancer (NSCLC) (n = 6), and pancreatic cancer (n = 1). Dose‐limiting toxicities occurred in six patients, including grade 3 diarrhea, dehydration, decreased appetite, nausea, vomiting, and mucositis. The recommended phase II dose (RP2D) was 20 mg afatinib once daily (QD) and 25 mg selumetinib b.i.d. (21 days on/7 days off) for continuous afatinib dosing and for intermittent dosing with both drugs 5 days on/2 days off. Efficacy was limited with disease stabilization for 221 days in a patient with NSCLC as best response.
Conclusion
Afatinib and selumetinib can be combined in continuous and intermittent schedules in patients with KRAS mutant tumors. Although target engagement was observed, the clinical efficacy was limited.
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