Improved Therapeutic Efficacy of Topotecan Against A549 Lung Cancer Cells with Folate-targeted Topotecan Liposomes.

BACKGROUND: Among all cancers, lung cancer has high mortality among patients in most of the countries in the world. Targeted delivery of anticancer drugs can significantly reduce the side effects and dramatically improve the effects of the treatment. Folate, a suitable ligand, can be modified to the surface of tumor-selective drug delivery systems because it can selectively bind to the folate receptor, which is highly expressed on the surface of lung tumor cells. OBJECTIVE: This study aimed to construct a kind of folate-targeted topotecan liposomes for investigating their efficacy and mechanism of action in the treatment of lung cancer in preclinical models. METHODS: We conjugated topotecan liposomes with folate, and the liposomes were characterized by particle size, entrapment efficiency, cytotoxicity to A549 cells and in vitro release profile. Technical evaluations were performed on lung cancer A549 cells and xenografted A549 cancer cells in female nude mice, and the pharmacokinetics of the drug were evaluated in female SD rats. RESULTS: The folate-targeted topotecan liposomes were proven to show effectiveness in targeting lung tumors. The anti-tumor effects of these liposomes were demonstrated by the decreased tumor volume and improved therapeutic efficacy. The folate-targeted topotecan liposomes also lengthened the topotecan blood circulation time. CONCLUSION: The folate-targeted topotecan liposomes are effective drug delivery systems and can be easily modified with folate, enabling the targeted liposomes to deliver topotecan to lung cancer cells and kill them, which could be used as potential carriers for lung chemotherapy.

Optimal Sampling Strategies for Irinotecan (CPT-11) and its Active Metabolite (SN-38) in Cancer Patients.

Irinotecan (CPT-11) is an anticancer agent widely used in the treatment of a variety of adult solid tumors. The objective of this study was to develop an optimal sampling strategy model that accurately estimates pharmacokinetic parameters of CPT-11 and its active metabolite, SN-38. This study included 221 patients with advanced solid tumors or lymphoma receiving CPT-11 single or combination therapy with 5-fluorouracil (5-FU)/leucovorin (LV) (FOLFIRI) plus bevacizumab from 4 separate clinical trials. Population pharmacokinetic analysis of CPT-11 and SN-38 was performed by non-linear mixed effects modeling. The optimal sampling strategy model was developed using D-optimality with expected distribution approach. The pharmacokinetic profiles of CPT-11 and SN-38 were best described by a 3- and 2-compartment model, respectively, with first-order elimination. Body surface area and co-administration with 5-FU/LV plus bevacizumab were significant covariates (p < 0.01) for volumes of the central compartment of CPT-11 and SN-38, and clearance of CPT-11. Pre-treatment total bilirubin and co-administration with 5-FU/LV and bevacizumab were significant covariates (p < 0.01) for clearance of SN-38. Accurate and precise predictive performance (r2 > 0.99, -2 < bias (%ME) < 0, precision (% RMSE) < 12) of both CPT-11 and SN-38 was achieved using: (i) 6 fixed sampling times collected at 1.5, 3.5, 4, 5.75, 22, 23.5 hours post-infusion; or (ii) 1 fixed time and 2 sampling windows collected at 1.5, [3-5.75], [22-23.5] hours post-infusion. The present study demonstrates that an optimal sampling design with three blood samples achieves accurate and precise pharmacokinetic parameter estimates for both CPT-11 and SN-38.

A fast method for the detection of irinotecan in plasma samples by combining solid phase extraction and differential pulse voltammetry.

In this paper, a fast method for the detection of irinotecan (CPT-11) in plasma samples was investigated. CPT-11 is widely used in a number of chemotherapeutic treatments of several solid tumors. The method is based on the combination of a solid phase extraction and an electrochemical detection step. The extraction of CPT-11 from plasma was performed using solid phase extraction (SPE) columns and acetonitrile as eluent. The procedure included also a cleaning step to eliminate interference due to plasma endogenous compounds and the co-therapeutics 5-fluoroacil (5-FU) and folinic acid (FA). The latter are administered together with CPT-11 in the FOLFIRI regimen. The detection of CPT-11 was performed by differential pulse voltammetry at a glassy carbon electrode (GCE) in basified acetonitrile media. Under these conditions, a well-defined peak due to the oxidation of the tertiary ammine end of CPT-11, also free from interference due to main metabolites, was obtained. Calibration plots showed a good linear response with limit of detection and quantification of 1.10 × 10-7 and 3.74 × 10-7 M, respectively. The suitability of the method proposed here for clinical applications was verified by determining the concentration of CPT-11 in plasma samples of an oncological patient, collected after 30 and 180 min from the infusion of the drug. Graphical abstract.

Pharmacometabolomics Reveals Irinotecan Mechanism of Action in Cancer Patients.

The purpose of this study was to identify early circulating metabolite changes implicated in the mechanism of action of irinotecan, a DNA topoisomerase I inhibitor, in cancer patients. A liquid chromatography-tandem mass spectrometry-based targeted metabolomic platform capable of measuring 254 endogenous metabolites was applied to profile circulating metabolites in plasma samples collected pre- and post-irinotecan treatment from 13 cancer patients. To gain further mechanistic insights, metabolic profiling was also performed for the culture medium of human primary hepatocytes (HepatoCells) and 2 cancer cell lines on exposure to SN-38 (an active metabolite of irinotecan). Intracellular reactive oxygen species (ROS) was detected by dihydroethidium assay. Irinotecan induced a global metabolic change in patient plasma, as represented by elevations of circulating purine/pyrimidine nucleobases, acylcarnitines, and specific amino acid metabolites. The plasma metabolic signature was well replicated in HepatoCells medium on SN-38 exposure, whereas in cancer cell medium SN-38 induced accumulation of pyrimidine/purine nucleosides and nucleobases while having no impact on acylcarnitines and amino acid metabolites. SN-38 induced ROS in HepatoCells, but not in cancer cells. Distinct metabolite signatures of SN-38 exposure in HepatoCells medium and cancer cell medium revealed different mechanisms of drug action on hepatocytes and cancer cells. Elevations in circulating purine/pyrimidine nucleobases may stem from nucleotide degradation following irinotecan-induced DNA double-strand breaks. Accumulations of circulating acylcarnitines and specific amino acid metabolites may reflect, at least in part, irinotecan-induced mitochondrial dysfunction and oxidative stress in the liver. The plasma metabolic signature of irinotecan exposure provides early insights into irinotecan mechanism of action in patients.

Pilot Study Comparing Systemic and Tissue Pharmacokinetics of Irinotecan and Metabolites after Hepatic Drug-Eluting Chemoembolization.

Differences in drug metabolism associated with UGT1A1 polymorphism could result in individualized local response to hepatic chemoembolization with irinotecan-eluting beads (DEBIRI) or predictable toxicities. Five patients with inoperable hepatic metastases from colorectal or anal malignancies treated with DEBIRI were assessed for UGT1A1 mutations. No difference in area under the curve (AUC) for SN38 in normal liver and tumor tissue samples was noted with variant or wild-type UBT1A1 (P = .16 and P = .05, respectively). Plasma SN-38 AUC was significantly lower in wild-type compared to variant patients (P < .0001). UGT1A1 genotype may not be predictive of hematologic toxicity after DEBIRI.

Dose adjustment of irinotecan based on UGT1A1 polymorphisms in patients with colorectal cancer.

PURPOSE: Irinotecan is effective for metastatic colorectal cancer (mCRC). SN-38 is an active metabolite of irinotecan, which is formed by carboxylesterase and inactivated by UDP-glucuronyltransferase (UGT) 1A1. The UGT enzyme activity is reduced in patients with homozygous mutation in UGT1A1 genes (*6/*6, *28/*28 and *6/*28); thus dose reduction is required for prevention of severe adverse events associated with irinotecan. The present study was designed to investigate the relationship between UGT1A1 polymorphisms and the incidence of adverse events or the therapeutic effect in mCRC patients who received irinotecan. METHODS: Sixty-three mCRC patients who received irinotecan during January 2014 and May 2018 were the subjects of this study. The incidence of adverse events, including diarrhea and neutropenia, and the therapeutic effect of irinotecan were compared among homozygous group, heterozygous group and wild-type group. The initial dose of irinotecan was 150 mg/m2 in the heterozygous group and wild-type group, while the dose was reduced by 20% (120 mg/m2) in the homozygous group. RESULTS: The UGT1A1 polymorphisms occurred in 15.9%, 33.3%, and 50.8% for homozygous group, heterozygous group, and wild-type group, respectively. The average dose of irinotecan during overall cycles was not significantly different among three groups, despite the reduction of initial dose in homozygous group. There were no significant differences in the incidence rates of adverse events, tumor response, or time to treatment failure among three groups. CONCLUSION: The present study demonstrated that dose reduction by 20% ensured safety and efficacy of irinotecan in mCRC patients with homozygous mutation in UGT1A1 genes.

Plasma pharmacokinetics of the indenoisoquinoline topoisomerase I inhibitor, NSC 743400, in rats and dogs.

PURPOSE: NSC 743400 is a novel synthetic indenoisoquinoline analog under development as an anticancer agent. It is a potent topoisomerase I inhibitor with potential therapeutic advantages over FDA-approved camptothecin derivatives. In preparation for clinical development of NSC 743400, we determined the pharmacokinetics after administration to rats and dogs. METHODS: NSC 743400 was administered intravenously at a dose of 12 or 24 mg/m(2) to rats (single bolus) or 10, 50, 100, 215, 430, or 646 mg/m(2) (intravenous infusion) or 860 or 1720 mg/m(2) (orally) to dogs. RESULTS: Intravenously administered NSC 743400 was eliminated from both species with an estimated t 1/2 of 2-5 h in rat and 6-14 h in dog. Elimination t 1/2 increased with dose in dog. Area under the plasma concentration-versus-time curve (AUC) was comparable in both species, at about 300-400 h ng/mL for the approximately 10 mg/m(2) dose groups. Overall, AUC values increased proportionally with dose for both species but had evidence of more than proportional exposure at the highest doses. Oral dosing resulted in variable drug absorption. CONCLUSIONS: The pharmacokinetic data were used to plan first-in-human clinical trials.

Integrative population pharmacokinetic and pharmacodynamic dose finding approach of the new camptothecin compound namitecan (ST1968).

AIMS: Namitecan is a new camptothecan compound undergoing early clinical development. This study was initiated to build an integrated pharmacokinetic (PK) and pharmacodynamic (PD) population model of namitecan to guide future clinical development. METHODS: Plasma concentration-time data, neutrophils and thrombocytes were pooled from two phase 1 studies in 90 patients with advanced solid tumours, receiving namitecan as a 2 h infusion on days 1 and 8 every 3 weeks (D1,8) (n = 34), once every 3 weeks (D1) (n = 29) and on 3 consecutive days (D1-3) (n = 27). A linear three compartment PK model was coupled to a semiphysiological PD-model for neutrophils and thrombocytes. Data simulations were used to interrogate various dosing regimens and give dosing recommendations. RESULTS: Clearance was estimated to be 0.15 l h(-1), with a long terminal half-life of 48 h. Body surface area was not associated with clearance, supporting flat-dosing of namitecan. A significant and clinically relevant association was found between namitecan area under the concentration-time curve (AUC) and the percentage drop of neutrophils (r(2) = 0.51, P < 10(-4)) or thrombocytes (r(2) = 0.49, P < 10(-4)). With a target for haematological dose-limiting toxicity of <20%, the recommended dose was defined as 12.5 mg for the D1,8 regimen, 23 mg for the once every 3 week regimen and 7 mg for the D1-3 regimen. CONCLUSION: This is the first integrated population PK-PD analysis of the new hydrophilic topoisomerase I inhibitor namitecan, that is currently undergoing early clinical development. A distinct relationship was found between drug exposure and haematological toxicity, supporting flat-dosing once every 3 weeks as the most adequate dosing regimen.

Insights into accelerated liposomal release of topotecan in plasma monitored by a non-invasive fluorescence spectroscopic method.

A non-invasive fluorescence method was developed to monitor liposomal release kinetics of the anticancer agent topotecan (TPT) in physiological fluids and subsequently used to explore the cause of accelerated release in plasma. Analyses of fluorescence excitation spectra confirmed that unencapsulated TPT exhibits a red shift in its spectrum as pH is increased. This property was used to monitor TPT release from actively loaded liposomal formulations having a low intravesicular pH. Mathematical release models were developed to extract reliable rate constants for TPT release in aqueous solutions monitored by fluorescence and release kinetics obtained by HPLC. Using the fluorescence method, accelerated TPT release was observed in plasma as previously reported in the literature. Simulations to estimate the intravesicular pH were conducted to demonstrate that accelerated release correlated with alterations in the low intravesicular pH. This was attributed to the presence of ammonia in plasma samples rather than proteins and other plasma components generally believed to alter release kinetics in physiological samples. These findings shed light on the critical role that ammonia may play in contributing to the preclinical/clinical variability and performance seen with actively-loaded liposomal formulations of TPT and other weakly-basic anticancer agents.

Irinotecan and temozolomide brain distribution: a focus on ABCB1.

Glioblastoma (GBM), the most common primary brain tumor in adults, is usually rapidly fatal with median survival duration of only 15 months and a 3-year survival rate of <7 %. Temozolomide (TMZ) is the only anticancer drug that has improved survival in GBM when administered with concomitant radiotherapy. Irinotecan (CPT-11) has also shown efficacy in recurrent gliomas monotherapy with moderate response. As the efficacy of GBM treatments relies on their brain distribution through the blood-brain barrier (BBB), the aim of the present work was to study, on an in vivo model, the brain distribution of TMZ, CPT-11 and its active metabolite, SN-38. We have focussed on the role of ABCB1, the main efflux transporter at the BBB level, through pharmacokinetics studies in CF1 mdr1a(+/+) and mdr1a(-/-) mice. Our results show that TMZ, CPT-11 and SN-38 are transported by ABCB1 at the BBB level with brain/plasma ratios of 1.1, 2.1 and 2.3, respectively.

PK/TD modeling for prediction of the effects of 8C2, an anti-topotecan mAb, on topotecan-induced toxicity in mice.

To facilitate the development of an inverse targeting strategy, where anti-topotecan antibodies are administered to prevent systemic toxicity following intraperitoneal topotecan, a pharmacokinetic/toxicodynamic (PK/TD) model was developed and evaluated. The pharmacokinetics of 8C2, a monoclonal anti-topotecan antibody, were assessed following IV and SC administration, and the data were characterized using a two compartmental model with nonlinear absorption and elimination. A hybrid PK model was constructed by combining a PBPK model for topotecan with the two-compartment model for 8C2, and the model was employed to predict the disposition of topotecan, 8C2, and the topotecan-8C2 complex. The model was linked to a toxicodynamic model for topotecan-induced weight-loss, and simulations were conducted to predict the effects of 8C2 on the toxicity of topotecan in mice. Increasing the molar dose ratio of 8C2 to topotecan resulted in a dose-dependent decrease in the unbound (i.e., not bound to 8C2) topotecan exposure in plasma (AUCf) and a decrease in the extent of topotecan-induced weight-loss. Consistent with model predictions, toxicodynamic experiments showed substantial reduction in the percent nadir weight loss observed with 30 mg/kg IP topotecan after co-administration of 8C2 (20 ± 8% vs. 10 ± 8%). The investigation supports the use of anti-topotecan mAb to reduce the systemic toxicity of IP topotecan chemotherapy.

Oral topotecan: Bioavailability, pharmacokinetics and impact of ABCG2 genotyping in Chinese patients with advanced cancers.

Oral topotecan (Hycamtin(®)) has been recently approved for the treatment of relapsed small cell lung cancer (SCLC) in 2007, however, the bioavailability and pharmacokinetic data of topotecan for Chinese patients is still limited. Xinze(®) is a new and the only capsule formulation of topotecan used in China that is similar to Hycamtin(®). The current study aimed to investigate the absolute bioavailability and pharmacokinetics of Xinze(®) in Chinese patients with advanced cancers. On day 1, an IV dose of 1.5 mg/m(2)/d as a 30 min continuous infusion was administered. Patients took the oral topotecan at one of two dose levels: 1.5 mg/m(2)/d (six patients) or 1.9 mg/m(2)/d (seven patients) on day 2. Plasma pharmacokinetics of total topotecan and topotecan in the lactone form were performed on both days using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Single-nucleotide polymorphisms (SNPs) identified in exon 5 (421C>A) and in exon 2 (34G>A) in ATP-binding cassette sub-family G member 2 (ABCG2) were analyzed by direct sequencing. Safety assessments were performed throughout the study. The maximum plasma concentration (Cmax) reached at 1-2 h and the elimination half-life time (T1/2) was approximately 4.2 h after oral administration. The absolute bioavailability of total topotecan in the 1.5 mg/m(2)/d and 1.9 mg/m(2)/d groups averaged 41.23 ± 11.8% and 36.00 ± 14.8%, respectively. The patients with heterozygous SNPs had essentially the same bioavailability and pharmacokinetics. The bioavailability of topotecan after oral administration illustrates good systemic exposure at dosages of 1.5 mg/m(2)/d and 1.9 mg/m(2)/d over a five-day schedule in Chinese patients. On a dose-normalized basis, the values of Cmax and AUC0-t for total topotecan in Chinese patients were higher than in Caucasians following oral and intravenous administration, while the T1/2 was consistent.

Development and validation of a liquid chromatography-tandem mass spectrometry method for topotecan determination in beagle dog plasma and its application in a bioequivalence study.

Topotecan (TPT) is an important anti-cancer drug that inhibits topoisomerase I. A sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that potentially determines TPT in beagle dog plasma is needed for a bioequivalence study of TPT formulations. We developed and validated LC-MS/MS to evaluate TPT in beagle dog plasma in terms of specificity, linearity, precision, accuracy, stability, extraction recovery and matrix effect. Plasma samples were treated with an Ostro(TM) sorbent plate (a robust and effective tool) to eliminate phospholipids and proteins before analysis. TPT and camptothecin (internal standard) were separated on an Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 50 mm) with 0.1% formic acid and methanol as the mobile phase at a flow rate of 0.25 mL/min. TPT was analyzed using positive ion electrospray ionization in multiple-reaction monitoring mode. The obtained lower limit of quantitation was 1 ng/mL (signal-to-noise ratio > 10). The standard calibration curve for TPT was linear (correlation coefficient > 0.99) at the concentration range of 1-400 ng/mL. The intra-day and inter-day precision, accuracy, stability, extraction recovery and matrix effect of TPT were within the acceptable limits. The validated method was successfully applied in a bioequivalence study of TPT in healthy beagle dogs.

Distribution of the anticancer drugs doxorubicin, mitoxantrone and topotecan in tumors and normal tissues.

PURPOSE: Pharmacokinetic analyses estimate the mean concentration of drug within a given tissue as a function of time, but do not give information about the spatial distribution of drugs within that tissue. Here, we compare the time-dependent spatial distribution of three anticancer drugs within tumors, heart, kidney, liver and brain. METHODS: Mice bearing various xenografts were treated with doxorubicin, mitoxantrone or topotecan. At various times after injection, tumors and samples of heart, kidney, liver and brain were excised. RESULTS: Within solid tumors, the distribution of doxorubicin, mitoxantrone and topotecan was limited to perivascular regions at 10 min after administration and the distance from blood vessels at which drug intensity fell to half was ~25-75 µm. Although drug distribution improved after 3 and 24 h, there remained a significant decrease in drug fluorescence with increasing distance from tumor blood vessels. Drug distribution was relatively uniform in the heart, kidney and liver with substantially greater perivascular drug uptake than in tumors. There was significantly higher total drug fluorescence in the liver than in tumors after 10 min, 3 and 24 h. Little to no drug fluorescence was observed in the brain. CONCLUSIONS: There are marked differences in the spatial distributions of three anticancer drugs within tumor tissue and normal tissues over time, with greater exposure to most normal tissues and limited drug distribution to many cells in tumors. Studies of the spatial distribution of drugs are required to complement pharmacokinetic data in order to better understand and predict drug effects and toxicities.

Sildenafil is not a useful modulator of ABCB1 and ABCG2 mediated drug resistance in vivo.

AIM: Recently, sildenafil was reported to be an inhibitor of P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in vitro. We have now investigated the in vivo potency of sildenafil. METHODS: By using wild-type and Abcb1; Abcg2 knockout mice we have investigated the effect of sildenafil on the brain penetration of two substrate drugs (docetaxel and topotecan). Next we have investigated if sildenafil was able to improve the efficacy of doxorubicin against P-glycoprotein expressing CT26 colon cancer cells in syngeneic Balb/c mice. RESULTS: Sildenafil administered orally at a dose of 50mg/kg did not improve the brain penetration of docetaxel and topotecan, although the plasma level of sildenafil was already much higher than can be achieved in humans. On the other hand, sildenafil increased the plasma levels of the cytotoxic drugs, but not by inhibition of Abcb1 or Abcg2, since this effect was also seen in Abcb1;Abcg2 knockout mice. The brain penetration of sildenafil was more than 20-fold higher in Abcb1;Abcg2 mice versus wild-type mice, indicating that sildenafil is a good substrate of the two transporters. Sildenafil was also not able to improve the efficacy of doxorubicin against subcutaneous CT26 tumours. The doxorubicin level in tumour tissue did increase, but so did the concentration of doxorubicin in plasma and heart. CONCLUSION: These results demonstrate that the potency and specificity of sildenafil as an inhibitor of ABCB1 and ABCG2 is not sufficient to warrant further clinical testing of this agent in combination with anticancer drugs.

Prolongation of time interval between doses could eliminate accelerated blood clearance phenomenon induced by pegylated liposomal topotecan.

Repeated injection of pegylated liposomes could elicit the disappearance of long-circulating characteristic, referred to as "accelerated blood clearance phenomenon." ABC phenomenon typically occurs when entrapped drugs are not cytotoxic, but recently it was reported that multiple doses of pegylated liposomal topotecan, a cytotoxic drug, could also induce the phenomenon in rats. To reveal whether the phenomenon could be induced in dogs and the effect of time interval between doses on the magnitude of ABC, pLT was repeatedly injected into beagle dogs with a time interval of 7, 21 and 28 days. The anti-PEG Ig M levels were detected using ELISA. It was found that ABC phenomenon could be induced in dogs by pLT. Inter-individual difference in anti-PEG antibody production could be observed, and antibody levels were directly correlated with the magnitude of ABC. Furthermore, time interval between doses had marked effect on the magnitude of ABC phenomenon. When the time interval was prolonged from 1 week to 4 weeks, ABC phenomenon could be eliminated. By comparing the pharmacokinetic profiles of lipid vesicles and entrapped topotecan, it was found that "empty pegylated vesicles" be formed in circulation, which might be responsible for the occurrence of ABC phenomenon.

Topotecan exposure estimation in pediatric acute myeloid leukemia supported by LC-MS-based drug monitoring and pharmacokinetic analysis.

Individualization of the topotecan dosing can reduce inter-patient variability, toxicity, and at the same time increases chemotherapy efficacy. Topotecan dosing based on simultaneous drug monitoring and pharmacokinetic analysis can yield more accurate and precise estimation of the topotecan systemic exposure than that attainable with the fixed dosing approach. Therefore, a combined approach could provide a tool assisting the clinicians in individualization of the topotecan dosing. The aim of the study was to estimate the topotecan exposure in pediatric patients with acute myeloid leukemia (AML) based on the plasma concentration-time data and using the pharmacokinetic analysis. The primary goal was achieve the correct estimation of the target plasma area against the topotecan concentration-time curve (AUC) in a 5 day course of cladribine followed by monitored topotecan in pediatric patients with recurrent/refractory AML. A sensitive and selective reversed-phase liquid chromatographic-mass spectrometry (LC-MS) assay was developed to quantify total topotecan in the human plasma samples. This method, with its lower quantification limit of 1 ng/ml, was validated over a linear range of 1-150 ng/ml. Under the proposed approach, the topotecan dosing was selected so as to achieve the final AUC value of 140±20 ng/ml h. The presented analytical and pharmacokinetic data demonstrate that the proposed approach can be a practical, useful, efficient, and accurate tool for individualizing the topotecan dosing in children with AML.

Topotecan vitreous and plasma levels and retinal toxicity after transcorneal intravitreal delivery in healthy albino rabbits: alternative retinoblastoma treatment.

AIM: To determine intravitreal and plasma concentrations and retinal toxicity after transcorneal intravitreal injection of 1 µg and 2 µg of topotecan (Hycamtin). METHOD: Twelve healthy albino rabbits were included in this in vivo experiment. Six anesthetized albino rabbits received a single transcorneal intravitreal injection of 1 µg (group A) or 2 µg (group B) of topotecan. Vitreous and blood samples were collected until 168 h. Left eyes were treated with the same volume of saline. Plasma and vitreous levels of topotecan were determined by high-performance liquid chromatography. Area under the plasma concentration time curve (AUC) was calculated using trapezoidal rule. Clinical evidence of toxicity was classified into four grades according to anatomical structures. Electroretinograms (ERGs) were recorded. RESULTS: Time to maximum concentration was observed up to 2 h after drug injection in group A whereas up to 1 h in group B. Low levels of topotecan were detected in plasma in both groups and in the vitreous humor of the contralateral eye in group B. Topotecan levels (mean vitreous AUC in group A 2.55 µg/mL.h and in group B 5.338 µg/mL.h) were detectable up to 6 h in both groups. We observed following structural changes in rabbit eyes: corneal vascularization, cataract, hemophthalmus, choroidal edema and focal retinal atrophy. Abnormal ERGs were obtained. CONCLUSION: Our findings proved that transcorneal intravitreal administration of 1 µg and 2 µg of topotecan results in potentially cytotoxic intraocular concentrations. More studies are needed to establish the safety of topotecan for retinoblastoma in children.

Pharmacokinetic modeling to assess factors affecting the oral bioavailability of the lactone and carboxylate forms of the lipophilic camptothecin analogue AR-67 in rats.

PURPOSE: Camptothecin analogues are anticancer drugs effective when dosed in protracted schedules. Such treatment is best suited for oral formulations. AR-67 is a novel lipophilic analogue with potent efficacy in preclinical models. Here we assessed factors that may influence its oral bioavailability in rats. METHODS: Plasma pharmacokinetic (PK) studies were conducted following administration of AR-67 lactone or carboxylate doses alone or after pre-dosing with inhibitors of the efflux transporters P-gp and Bcrp. A population PK model that simultaneously fitted to oral and intravenous data was used to estimate the bioavailability (F) and clearance of AR-67. RESULTS: An inverse Gaussian function was used as the oral input into the model and provided the best fits. Covariate analysis showed that the bioavailability of the lactone, but not its clearance, was dose dependent. Consistent with this observation, the bioavailability of AR-67 increased when animals were pretreated orally with GF120918 or Zosuquidar. CONCLUSION: Absorption of AR-67 is likely affected by solubility of its lactone form and interaction with efflux pumps in the gut. AR-67 appears to be absorbed as the lactone form, most likely due to gastric pH favoring its formation and predominance. F increased at higher doses suggesting saturation of efflux mechanisms.

Method development and validation of the simultaneous determination of a novel topoisomerase 1 inhibitor, the prodrug, and the active metabolite in human plasma using column-switching LC-MS/MS, and its application in a clinical trial.

A robust and sensitive method using liquid chromatography-tandem mass spectrometry was developed and validated for the simultaneous determination of a novel topoisomerase 1 inhibitor CH0793076 (3076), the prodrug CH4556300 (TP300), and the active metabolite CH0793011 (3011) in human plasma. All plasma analyzed with this method was acidified with 1M HCl and 46% citric acid solution in a ratio of 100:10:1 (v:v:v) to avoid the pH-based degradation of TP300 and to shift the equilibria of 3076 and 3011 between the lactone and carboxylate forms towards the lactone forms. After the plasma proteins were precipitated with methanol:acetonitrile:HCl 1M (50:50:1, v:v:v) containing stable isotopic internal standards, the analytes were trapped on an Xterra MS C18 column (10×2.1 mm i.d., 5 µm) and separated on a Gemini C18 column (50×2.0 mm i.d., 5 µm) using column-switching liquid chromatography. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to quantify the analytes with transitions m/z 587.2>441.2 for TP300, 459.1>415.2 for 3076, and 475.1>361.1 for 3011. The inter- and intra-day precisions were below 12%, and the accuracy was between -16% and 16% at the lower limit of quantitation (LLOQ) and between -11% and 14% at the other quality controls. The LLOQs of TP300, 3076, and 3011 were 0.8, 0.04, and 0.04 ng/mL, respectively. The validated method was successfully applied to clinical sample analysis and incurred sample reanalysis was also conducted.