Drug Levels In Tissues Research Articles

Do albendazole-loaded lipid nanocapsules enhance the bioavailability of albendazole in the brain of healthy mice?

Neurocysticercosis is a neglected tropical disease that affects the central nervous system and is the most common cause of human epilepsy acquired in developing countries. Therapeutic failures attributed to medical management of neurocysticercosis with albendazole (ABZ) have been primarily linked to the poor drug absorption rate resulting in low drug level in plasma and brain tissue. The aim of the current work was to characterize and compare the brain pharmacokinetic behavior of ABZ formulated as a suspension or lipid nanocapsules (ABZ-LNCs) in healthy mice. The relative availability in brain tissue of the active metabolite ABZ sulphoxide increased 183% when ABZ was administered as LNCs, in relation to ABZ suspension. The parent drug was also detected for a short period of time. The bioavailability of ABZ in ABZ-LNCs treated mice increased more than 2 fold compared with ABZ suspension group. The enhanced drug brain exposure observed after administration of ABZ-LNCs to healthy mice has potential usefulness for the treatment of human neurocysticercosis.

P1.14-20 Tarloxotinib as a Novel Therapeutic Strategy for Oncogenic Alterations Across the ErbB Family of Receptors

The ErbB family of receptors tyrosine kinases (EGFR, HER2, HER3, and HER4) have been implicated in multiple different tumor types. The implementation of comprehensive next generation sequencing has allowed the identification of diverse gene alterations that function as oncogene drivers in these receptors. Some of the non-common gene alterations identified are resistant to marketed EGFR/HER2 inhibitors. Tarloxotinib is a prodrug that generates a potent and irreversible pan-HER inhibitor (tarloxotinib-E) under hypoxic conditions associated with tumors. In this study we evaluated the effect of tarloxotinib on several types of oncogenic mutations and fusions that involve the ErbB family of receptors. cDNAs encoding EGFR kinase domain duplications (EGFR-EGFR), EGFR-RAD51 gene fusion, EGFR-ERBB4, ERBB2-GRB7 and EZR-ERBB4 gene fusion were expressed in Ba/F3 cells. Using spheroid assays we evaluated the proliferation of A172 glioblastoma cell line treated with tarloxotinib, tarloxotinib-E or 1st, 2ndor 3rdgeneration EGFR/HER2 TKIs. We analyze the on target and signaling effects elicited by tarloxotinib-E via immunoblots. Using a nude mice xenograft model of the human derived cell line CUTO17 wi the EGFR exon 20 insertion p.N771_H773dupNPH, we evaluated tumor, tissue and blood drug levels by mass spectrometry and the effect of tarloxotinib on tumor growth. Our results demonstrate that tarloxotinib-E inhibits phosphorylation of EGFR with a kinase duplication and inhibits proliferation in a spheroid invasion assay in A172 cells. In the CUTO17 EGFR exon 20 model, treatment with tarloxotinib inhibited tumor growth. Intratumor levels of tarloxotinib-E were ∼20 times higher than skin and ∼50 times higher than plasma demonstrating selective tumor conversion of tarloxotinib. Cell growth inhibition (EC50) of novel HER family fusions (EGFR-EGFR, EGFR-RAD51, EGFR-ERBB4, ERRB2-GRB7 and EZR-ERBB4) will be presented. Tarloxotinib is a potent irreversible inhibitor in vitro for cells that harbor oncogenic alterations across the ERBB gene family, including EGFR kinase domain duplications, ErbB fusions and exon-20 insertions. Tarloxotinib is selectively activated in hypoxic tumor regions demonstrating a novel mechanism to generate a therapeutic window and avoid on-target EGFR-related toxicities.

Biological Obstacles for Identifying In Vitro-In Vivo Correlations of Orally Inhaled Formulations.

Oral inhalation of drugs is the classic therapy of obstructive lung diseases. In contrast to the oral route, the link between in vitro and in vivo findings is less well defined and predictive models and parameters for in vitro-in vivo correlations are missing. Frequently used in vitro models and problems in obtaining in vivo values to establish such models and to identify the action of formulations in vivo are discussed. It may be concluded that major obstacles to link in vitro parameters on in vivo action include lack of treatment adherence and incorrect use of inhalers by patients, variation in inhaler performance, changes by humidity, uncertainties about lung deposition, and difficulties to measure drug levels in epithelial lining fluid and tissue. Physiologically more relevant in vitro models, improvement in inhaler performance, and better techniques for in vivo measurements may help to better understand importance and interactions between individual in vitro parameters in pulmonary delivery.

Abstract 1136: Tumor uptake and biodistribution of 89Zirconium-labeled ipilimumab in patients with metastatic melanoma during ipilimumab treatment

Abstract Introduction Ipilimumab, a monoclonal antibody targeting CTLA-4, is approved for the treatment of metastatic melanoma and significantly improves overall survival. Because of the high costs and the potential serious toxicity of ipilimumab, it is of great importance to identify biomarkers that correlate with clinical activity and that can be used to select patients who will benefit from CTLA-4 blockade therapy. We hypothesize that patients who do not respond to treatment with ipilimumab have lower drug levels in tumor tissues as compared to patients with a good response to therapy. In addition, we hypothesize that immune related adverse events (irAEs) are associated with high drug levels in the affected tissue. As irAEs usually occur approximately 6-8 weeks after the first injection of ipilimumab, we hypothesize that the drug levels in potentially affected tissues will increase at the second injection. Experimental procedures To visualize in vivo localization of ipilimumab in patients diagnosed with metastatic melanoma, 37 MBq, 10 mg 89Zr-labeled ipilimumab was injected within 2 hours after their first ipilimumab dose (3 mg/kg). Whole body PET/CT scans were obtained at 2h, 72h and 144h post injection and this procedure was repeated three weeks later at the second ipilimumab cycle. Biodistribution and tumor uptake were assessed visually by a nuclear physician. Focal uptake in tumor lesions exceeding local background was determined in volumes of interest (VOI) and SUVpeak values were obtained. Biodistribution was quantified by defining vital organs (i.e. lungs, kidneys, spleen, liver) and calculating mean %ID/kg. Blood was drawn for dosimetry and immunophenotyping at several time points during the trial. Presented here are initial results of the first three patients, up to 29 patients are planned to be included. Results Biodistribution of 89Zr-labeled ipilimumab showed a pattern distinctive for 89Zr-labeled antibodies with uptake in liver and spleen, as well as prolonged circulating antibody in the bloodstream corresponding to the pharmacokinetics of ipilimumab. Visual evaluation confirmed uptake of 89Zr-labeled ipilimumab in 5/12 evaluable tumor lesions, visible at both first and second injection of ipilimumab. Tumor uptake was comparable for 72h and 144h post injection with a mean of 6.9 %ID/kg (range 3.3-10.1) and a SUVpeak of 4.4 (range 2.3-8.9). There were no significant differences in tumor uptake between first and second dose of ipilimumab (mean 7.31 and 6.54 %ID/kg respectively). Conclusions Preliminary data of this ongoing study showed that the tracer is able to visualize and quantify uptake of ipilimumab in tumors. Correlations between tumor uptake and response to treatment will be presented. Furthermore, special interest will be given to uptake in lymphoid organs and locations for irAEs. Citation Format: Iris H. Miedema, Gerben J. Zwezerijnen, Guus A. van Dongen, Daniëlle J. Vugts, Marc C. Huisman, Otto S. Hoekstra, Tanja D. de Gruijl, Hendrik M. Verheul, Catharina W. Menke, Alfons J. van den Eertwegh. Tumor uptake and biodistribution of 89Zirconium-labeled ipilimumab in patients with metastatic melanoma during ipilimumab treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1136.

Lipid-Based Nanoparticles: Application and Recent Advances in Cancer Treatment.

Many therapeutically active molecules are non-soluble in aqueous systems, chemically and biologically fragile or present severe side effects. Lipid-based nanoparticle (LBNP) systems represent one of the most promising colloidal carriers for bioactive organic molecules. Their current application in oncology has revolutionized cancer treatment by improving the antitumor activity of several chemotherapeutic agents. LBNPs advantages include high temporal and thermal stability, high loading capacity, ease of preparation, low production costs, and large-scale industrial production since they can be prepared from natural sources. Moreover, the association of chemotherapeutic agents with lipid nanoparticles reduces active therapeutic dose and toxicity, decreases drug resistance and increases drug levels in tumor tissue by decreasing them in healthy tissue. LBNPs have been extensively assayed in in vitro cancer therapy but also in vivo, with promising results in some clinical trials. This review summarizes the types of LBNPs that have been developed in recent years and the main results when applied in cancer treatment, including essential assays in patients.

Review on Targeted Drug Delivery Carriers Used in Nanobiomedical Applications

Targeted drug delivery (TDD) is an advanced and smart method of delivering drugs to the patients in a targeted sequence that increases the concentration of delivered drug only at the targeted body part of interest (organs/tissues/cells). This will in turn enhance efficacy of treatment by reducing side effects and the required dose of the drug. TDD ensures a certain defined minimally required constant amount of a therapeutic agent for a prolonged period of time to a targeted diseased area within the body. This helps maintain the required plasma and tissue drug levels in the body thereby avoiding any damage to the healthy tissue via the drug. Various drug carriers that are envisaged in advanced delivery systems are soluble polymers, inorganic nanoparticles, magnetic nanoparticles, biodegradable microsphere polymers (synthetic and natural), neutrophils, fibroblasts, artificial cells, lipoproteins, liposomes, micelles and immune micelle. In selecting such a vehicle, important factors to consider are chemical and physical properties drugs, side effects or cytotoxicity to healthy cells, route to be taken for the delivery of the drug, the targeted site, and the disease. As such, TDD formulations are prepared by considering the specific properties of target cells, nature of markers or transport carriers or vehicles, which convey drug to specific receptors, and ligands and physically modulated components.

Abstract OT1-04-01: A phase IIB pre-surgical trial of oral tamoxifen (TAM) versus transdermal 4-hydroxytamoxifen (4-OHT) in women with DCIS of the breast

Abstract Background Ductal carcinoma in situ (DCIS) is diagnosed in 60,000 women annually in the US. TAM is proven to reduce risk of local recurrence and new primary breast cancer in women with estrogen receptor (ER) positive DCIS. However, acceptance of TAM has been low, primarily because of toxicity related to systemic exposure. Local delivery to the breast is an attractive alternative since low systemic levels could minimize toxicity. 4-OHT is an active metabolite of TAM. When formulated as a gel and applied to the breast skin, it is well tolerated, and results in 4-OHT breast tissue drug levels comparable oral TAM. In small pilot studies, its anti-proliferative effects on invasive breast tumors and DCIS are also similar to oral TAM [Lee O, et al. PMID 25028506]. The goal of our study is to validate these results in preparation for a Phase III trial of 4-OHT gel in comparison to oral TAM. Methods We are conducting a randomized, double-blinded, placebo-controlled, Phase IIB pre-surgical trial to demonstrate that daily application of 4-OHT gel will result in a reduction in the Ki-67 labeling index of DCIS lesions that is not inferior to that seen in women receiving daily oral TAM 20 mg daily. Ki-67 of the base-line diagnostic core needle biopsy will be compared to that of the therapeutic surgical excision sample after oral TAM or 4-OHT gel for 8 ± 2 weeks. Secondary endpoints include changes in Oncotype DCIS-Score, IHC markers (CD68, COX2, p16), hormone levels, coagulation markers, drug concentration in the plasma and breast tissue, the fraction of women with no residual DCIS in the surgical sample, and experienced symptoms. 100 women (assuming 20% non-evaluable samples or compliance issues) with DCIS (10% ER-positive) will be enrolled across six institutions into two intervention arms: oral TAM 20 mg daily, placebo gel and 4-OHT gel 4mg daily (2mg/breast), placebo capsule. All participants will be evaluable for toxicity from their first dose. All samples from all participants who receive drug will be evaluated and included in the primary analysis, which will be based on intent to treat principle. To date 15 of 100 participants have been enrolled across six institutions including: Northwestern University in Chicago, IL, St. Elizabeth Healthcare in Edgewood, KY, Duke University Medical Center in Durham, NC, Cleveland Clinic in Cleveland, OH, Memorial Sloan Kettering Cancer Center in New York, NY, and Mayo Clinic in Rochester, MN. Since study open, 69 potential participants have been contacted, 52 did not consent for screening, 17consented for screening, 2 are pending consent, and 15 have started study intervention. The most common reasons potential participants chose not to consent are wanting to schedule surgery as soon as possible, attitudes toward medical research, and current use of a prohibited concomitant medication such as a potent inhibitor of tamoxifen metabolism or exogenous sex steroid. Funding Source: NCI Contract # HHSN2612201200035I. Citation Format: Benante KA, Xu Y, Tull MB, Segura AJ, Alber KM, Kalinichenko K, Hou L, Jovanovic B, Perloff M, Heckman-Stoddard B, Dimond E, Khan SA. A phase IIB pre-surgical trial of oral tamoxifen (TAM) versus transdermal 4-hydroxytamoxifen (4-OHT) in women with DCIS of the breast [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-04-01.

Intracameral Sustained-Release Bimatoprost Implant Delivers Bimatoprost to Target Tissues with Reduced Drug Exposure to Off-Target Tissues.

Purpose: To explore the ocular distribution of bimatoprost after intracameral administration of a biodegradable sustained-release bimatoprost implant (Bimatoprost SR) versus repeated topical administration of bimatoprost 0.03% ophthalmic solution in dogs. Bimatoprost SR and topical bimatoprost 0.03% previously were shown to have similar intraocular pressure-lowering effects in humans in a phase 1/2 clinical trial.Methods: Twenty-four beagle dogs received either once-daily topical bimatoprost 0.03% for 7 days or a bilateral intracameral administration of Bimatoprost SR (15 μg). At predetermined time points, ocular tissues were collected and concentrations of bimatoprost and bimatoprost acid were quantified using liquid chromatography–tandem mass spectrometry.Results: Bimatoprost SR administration enhanced delivery of study drug to a site of action [iris–ciliary body (ICB)] compared with topical bimatoprost (Cmax [bimatoprost+bimatoprost acid] = 18,200 and 4.13 ng/g, respectively). However, distribution of drug to tissues associated with prostaglandin analog (PGA)-related side effects (i.e., bulbar conjunctiva, eyelid margins, and periorbital fat) was limited following Bimatoprost SR administration (Cmax [bimatoprost+bimatoprost acid] = BLQ [beneath the limit of quantitation] to 0.354 ng/g) compared with topical dosing (Cmax [bimatoprost+bimatoprost acid] = 36.6–2,110 ng/g).Conclusions: Bimatoprost SR administration in dogs selectively delivered drug to the ICB with low or undetectable drug levels in ocular surface and extraocular tissues. Use of Bimatoprost SR for glaucoma treatment may reduce the incidence of adverse events typically associated with topical PGAs by targeting bimatoprost delivery to the key site of action of the PGA class and reducing exposure to off-target tissues.

In Vivo Evaluation of Novel Geranylgeranyl Diphosphate Synthase Inhibitors

▪The enzyme geranylgeranyl diphosphate synthase (GGDPS) synthesizes the 20-carbon isoprenoid geranylgeranyl diphosphate which is used in protein geranylgeranylation reactions. Our work has demonstrated that GGDPS inhibitors (GGDPSIs) represent a novel therapeutic strategy for multiple myeloma (MM) by disrupting Rab protein geranylgeranylation. Treatment of MM cells with GGDPSI results in disruption of monoclonal protein trafficking, leading to induction of the unfolded protein response pathway (UPR) and apoptosis. We have previously reported preclinical studies with a lead GGDPSI, VSW1198 (a mixture of homogeranyl/homoneryl triazole bisphosphonates), demonstrating the agent's metabolic stability, prolonged half-life (plasma elimination half-life of 47.7 (±7.4) hrs), systemic distribution and confirmed in vivo disruption of geranylgeranylation (Haney et al., Invest New Drugs, 2018). Additional structure-function efforts have led to the development of the α-methylated derivatives RAM2093 (homogeranyl) and RAM2061 (homoneryl). Intriguingly the addition of the α-methyl group abrogates the effects of the olefin stereochemistry on inhibitor potency such that the individual isomers display near identical ability to disrupt protein geranylgeranylation in enzyme and cell assays (Matthiesen et al., Bioorg Med Chem, 2018). As little is known regarding the impact of olefin stereochemistry on the pharmacokinetic (PK)/pharmacodynamic (PD) properties of drugs, we pursued additional in vitro and in vivo studies of RAM2093 and RAM2061 and investigated the efficacy of the GGDPSIs in a mouse MM xenograft model. In MM cell lines, qRT-PCR and western blot analysis showed that both isomers induce activation of UPR/apoptotic markers in a dose-dependent manner and with similar potency. Single dose testing in CD-1 mice identified a maximum tolerated dose of 0.5 mg/kg IV for RAM2061 and 0.3 mg/kg for RAM2093. Liver toxicity was the primary barrier to dose escalation with both compounds. Analysis of blood samples showed elevated liver transaminase levels with normal bilirubin/alkaline phosphatase levels and histopathological examination confirmed evidence of hepatocyte injury at higher doses. Multi-dose schedules of 0.1 mg/kg twice a week (two weeks on, one week off, two weeks on) for RAM2061 and RAM2093, as well as 0.2 mg/kg (RAM2061) and 0.15 mg/kg (RAM2093) weekly x four weeks were tested. All dosing schedules were tolerated with the exception of the twice-weekly schedule for RAM2093. Consistent with the findings from our single dose testing, both multi-dose schedules induced transient elevation of hepatic transaminases. No loss of weight was observed and creatinine and CBC results were within normal limits for both single and multi-dose injected animals. Importantly, western blot analysis of mouse tissues collected from both RAM2061 and RAM2093 multi-dose-treated mice showed accumulation of unmodified Rap1a in the liver, kidney and spleen, indicating in vivo disruption of protein geranylgeranylation was achieved. Furthermore, using MM.1S MM cell flank xenographs in NOD-SCID mice, we observed a significant reduction in tumor growth in mice treated with either VSW1198 or RAM2061 relative to vehicle control. Lastly, PK/biodistribution studies with RAM2093 and RAM2061 were performed following a single dose of 0.3 mg/kg IV. Both compounds were detectable in plasma and liver samples for up to seven days post-injection demonstrating prolonged half-life and tissue distribution. The full PK parameters for both compounds will be presented and studies measuring drug levels in other tissues are ongoing. Taken together, these data suggest that RAM2061 and RAM2093 have equivalent anti-MM activity in vitro, but that RAM2061 is better tolerated in vivo. Whether this is a consequence of different PK properties vs. differences in tissue uptake or metabolism will be determined. These studies also confirm the in vivo efficacy of our novel GGDPSIs and support further development of these agents for the treatment of MM. [Display omitted] DisclosuresHolstein:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Safety of Endoscopic-Ultrasound-Guided Portal Injection Chemotherapy using Drug-Eluting Microbeads in a Porcine Model

Background and Aims: Patients with diffuse liver metastases have systemic chemotherapy as their only treatment option. We developed Endoscopic Ultrasound (EUS)-guided portal injection chemotherapy (EPIC) to increase drug levels in hepatic tissue as a novel new liver directed therapy. Methods: Sixteen anesthetized pigs were treated with 50 mg of irinotecan (n=8) or doxorubicin (n=8). Half (n=4) of the animals in each drug group were treated with EPIC-injected microbeads or EUS-guided chemotherapy without beads into the inferior vena cava (control). Animals were observed twice daily for 7 days for signs of clinical toxicities. Tissue samples were harvested for histology and drug levels. Blood counts and chemistries were determined pre-treatment and at 7 days. Results: No toxicities as evidenced by abnormal animal behavior were observed. No significant changes occurred in blood chemistry or blood counts in the irinotecan groups. For doxorubicin, systemic injection significantly decreased albumin, hemoglobin, and white blood cell count (P<.05), with no changes after EPIC. Hepatic histology showed mild foreign body reactions around the beads. No significant histologic changes were seen in other tissue sites. Neither irinotecan nor SN-38 was detectable at 7 days. For doxorubicin, no drug was detected in the plasma or bone marrow. The mean (SD) doxorubicin hepatic levels were non-significantly increased with EPIC vs control (181 [241] vs 151 [67] ng/g). Cardiac doxorubicin levels were significantly lower with EPIC (15 [4] vs 138 [48] ng/g; P=.02). Conclusions: EPIC using drug-eluting microbeads was safe in this animal model. For doxorubicin, EPIC may be safer than systemic injection.

Noncovalent PEG Coating of Nanoparticle Drug Carriers Improves the Local Pharmacokinetics of Rectal Anti-HIV Microbicides.

Antiretroviral drug nanocarriers hold great promise for developing anti-human immunodeficiency virus (HIV) rectal microbicides. However, challenges remain, namely, concerning which properties are more suited for enhancing colorectal distribution and retention of microbicide compounds. In this work, we developed and assessed the in vitro and in vivo performance of poly(lactic- co-glycolic acid) (PLGA)-based nanoparticles (NPs) as carriers for the model drug efavirenz (EFV). We particularly focused on the effect of noncovalent poly(ethylene glycol) coating of PLGA NPs (PEG-PLGA NPs) conferring a mucus-diffusive behavior on the pharmacokinetics (PK) of EFV following rectal administration to mice. Drug-loaded PLGA NPs and PEG-PLGA NPs (200-225 nm) were obtained by nanoprecipitation. Both types of systems were able to retain native antiretroviral activity of EFV in vitro, while featuring lower cytotoxicity against different epithelial cell lines and HIV target cells. Also, PLGA NPs and PEG-PLGA NPs were readily taken up by colorectal cell lines and mildly reduced EFV permeation while increasing membrane retention in Caco-2 and Caco-2/HT29-MTX cell monolayer models. When administered intrarectally to CD-1 mice in phosphate-buffered saline (pH 7.4), EFV-loaded PEG-PLGA NPs consistently provided higher drug levels in colorectal tissues and lavages, as compared to free EFV or drug-loaded PLGA NPs. Mean values for the area-under-the-curve between 15 min and 12 h following administration were particularly higher for PEG-PLGA NPs in distal and middle colorectal tissues, with relative bioavailability values of 3.7 and 29, respectively, as compared to free EFV (2.2 and 6.0 over PLGA NPs, respectively). Systemic exposure to EFV was reduced for all treatments. NPs were further shown safe after once-daily administration for 14 days, as assessed by histological analysis of colorectal tissues and chemokine/cytokine assay of rectal lavages. Overall, PEG-PLGA NPs demonstrated to be safe carriers for rectal microbicide drug delivery and able to provide enhanced local PK that could be valuable in preventing rectal HIV transmission.

Continuous low-dose infusion of patupilone increases the therapeutic index in mouse and rat tumour models.

Patupilone is a microtubule-targeted cytotoxic agent with clinical efficacy, but causes diarrhoea in more than 80% of patients. The efficacy and tolerability of patupilone delivered continuously by subcutaneous (s.c.) mini-pumps [(mini-pump dose (MPD)] or by intravenous bolus administration [intravenous bolus dose (IVBD)] were compared preclinically to determine whether the therapeutic index could be improved. The antiproliferative potency in vitro of patupilone was determined by measuring total cell protein. Tumours were grown s.c. in rats (A15) or nude mice (KB31, KB8511) or intracranially in nude mice (NCI-H460-Luc). Efficacy was monitored by measuring tumour volumes, bioluminescence or survival. Toxicity was monitored by body weight and/or diarrhoea. Total drug levels in blood, plasma, tissues or dialysates were quantified ex-vivo by liquid chromatography-mass spectroscopy/mass spectroscopy. Patupilone was potent in vitro with GI50s of 0.24-0.28 nmol/l and GI90s of 0.46-1.64 nmol/l. In rats, a single IVBD of patupilone dose dependently inhibited the growth of A15 tumours, but also caused dose-dependent body weight loss and diarrhoea, whereas MPD achieved similar efficacy, but no toxicity. In mice, MPD showed efficacy similar to that of IVBD against KB31 and KB8511 tumours, but with reduced toxicity. In a mouse intracranial tumour model, IVBD was more efficacious than MPD, consistent with patupilone concentrations in the brain. MPD provided constant plasma levels, whereas IVBD had very high C0/Cmin ratios of 70-280 (rat) or 8000 (mouse) over the dosing cycle. Overall, the correlation of plasma and tumour levels with response indicated that a Cave of at least GI90 led to tumour stasis. Continuous low concentrations of patupilone by MPD increased the therapeutic index in s.c. rodent tumour models compared with IVBD by maintaining efficacy, but reducing toxicity.

Ciclesonide and budesonide suspensions for nebulization delivery: An in vivo inhalation biopharmaceutics investigation

The pulmonary fate of inhaled poorly water-soluble drugs is not entirely clear. In this study, the main objective was to investigate the in vivo inhalation biopharmaceutics in the aspects of dissolution, mucociliary clearance, absorption and tissue binding using intratracheally administered budesonide and ciclesonide suspensions as model drugs. In doing so, this study first developed a method to differentiate between dissolved and undissolved ciclesonide in the lungs for evaluating in vivo dissolution. Following deposited in rat airways, the drug particles underwent rapid dissolution and mucociliary clearance, leading to the complete removal of drugs from the airways within 2 h and a limited absorption time less than 2 h. Upon dissolution, budesonide and ciclesonide were taken up and retained in the lung tissues for up to 12 h and 24 h, respectively. The in vivo dissolution profiles in the airways exhibited the sameness as the in vitro counterparts in a 0.5% sodium dodecyl sulfate solution as indicated by the similarity factor f2. The efficacy results in a lipopolysaccharide induced lung injury model showed that the duration of local anti-inflammatory was dependent on the drug levels in the lung tissues, but not on the in vitro/in vivo dissolution and plasma pharmacokinetics. The present results demonstrated that ciclesonide suspension has the potential to achieve once-daily dosing for nebulization therapy and the in vitro dissolution profile has limited usefulness in predicting in vitro–in vivo correlation.

Pranoprofen quantification in ex vivo corneal and scleral permeation samples: Analytical validation

The investigation of the ocular permeability and/or distribution of pranoprofen (PF), a non-steroidal antiinflamatory drug, demands for the selective analysis of its transit through specific ocular membranes. Therefore, customised ex vivo permeation experiments through external ocular tissues (cornea and sclera) have been validated for this drug in addition to its HPLC-UV quantification following standard bioanalytical guidelines. Chromatographic conditions consist of an isocratic system to elute the drug with a C18 column with UV detection at 245 nm. Precision, expressed as the relative standard deviation (% RSD), ranged between 4.89 and 0.79% (intra-day) and between 9.02 and 2.14% (interday). Accuracy ranged between 5.15 and -1.92% in intra-day experiments and between 6.25 and −4.89% in inter-day experiments. Drug recovery from tissue samples was reproducible around 90% and considered satisfactory to adequately assess drug levels in target tissues. Results indicate that the procedure is valid for the quantitation of PF in those ophthalmic samples in the range of 6.5 μg/mL to 100 μg/ml. As a proof of concept, PF permeation profiles through porcine cornea and sclera with vertical diffusion cells have been generated and analyzed. Pilot experiments demonstrate its applicability to investigate permeation levels of PF from 22.31 μg/cm2 (about a 20% of the dose) until 500 μg/cm2 if required. Additionally, real tissue-retention samples were also generated to verify the goodness of this experimental setup.

PREPARATION, CHARACTERISATION AND EVALUATION OF ROPINIROLE HYDROCHLORIDE LOADED CONTROLLED RELEASE MICROSPHERES USING SOLVENT EVAPORATION TECHNIQUE

Objective: The major objective of the research work was to design, characterise and evaluate controlled release microspheres of ropinirole hydrochloride by using non-aqueous solvent evaporation technique to facilitate the delivery of the drug at a predetermined rate for a specific period of time.Methods: Ropinirole hydrochloride microspheres were prepared by using different low-density polymers such as eudragit RL 100, eudragit RS 100 and ethylcellulose either alone or in combination with the help of non-aqueous solvent evaporation technique. All the formulated microparticles were subjected to various evaluation parameters such as particle size analysis, micrometric properties, drug entrapment efficiency, percentage drug loading, percentage yield and in vitro drug release study. The compatibility of the drug and polymers was confirmed by physical compatibility study, fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and x-ray diffraction study (XRD). The formation of the most optimized batch of the microsphere (F12) was confirmed by scanning electron microscopy (SEM), DSC, FTIR, and XRD. In vitro drug release study and in vitro drug release kinetics study of the formulated microspheres were also carried out.Results: Drug-polymer compatibility studies performed with the help of FTIR and DSC indicated that there were no interactions. Results revealed that non-aqueous solvent evaporation technique was a suitable technique for the preparation of microspheres as most of the formulations were discrete, free-flowing and spherical in shape with a good yield of 55.67% to 80.09%, percentage drug loading of 35.52% to 94.50% and percentage drug entrapment efficiency of 36.24% to 95.07%. Different drug-polymer ratios, as well as the combination of polymers, played a significant role in the variation of over-all characteristics of formulations. Based on the data of various evaluation parameters such as particle size analysis, percentage drug loading, percentage drug entrapment, percentage yield, rheological studies and in vitro drug release characteristics, formulation F12 was found to fulfil the criteria of ideal controlled release drug delivery system. F12 showed controlled release till the 14th hour (97.99%) and its in vitro release kinetics was best explained by zero-order kinetics and followed Korsemeyer-Pappas model (Non-Fickian mechanism). SEM of F12 revealed the formation of spherical structures. The FTIR study of F12 confirmed the stable nature of ropinirole in the drug-loaded microspheres. DSC and XRD patterns showed that ropinirole hydrochloride was dispersed at the molecular level in the polymer matrix.Conclusion: The controlled release microparticles were successfully prepared and from this study, it was concluded that the developed microspheres of ropinirole hydrochloride can be used for controlled drug release to improve the bioavailability and patient compliance and to maintain a constant drug level in the blood target tissue by releasing the drug in zero order pattern.

Hypocrellin B-loaded, folate-conjugated polymeric micelle for intraperitoneal targeting of ovarian cancer invitro and invivo.

Photodynamic therapy (PDT) is considered an innovative and attractive modality to treat ovarian cancer. In the present study, a biodegradable polymer poly (ethylene glycol) (PEG)‐poly (lactic acid)(PLA)‐folate (FA‐PEG‐PLA) was prepared in order to synthesize an active‐targeting, water‐soluble and pharmacomodulated photosensitizer nanocarrier. Drug‐loading content, encapsulation efficiency, in vitro and in vivo release were characterized, in which hypocrellin B (HB)/FA‐PEG‐PLA micelles had a high encapsulation efficiency and much slower control release for drugs compared to free drugs (P < .05). To evaluate the targeting ability of the HB/FA‐PEG‐PLA micelles, a cellular uptake study in vitro was carried out, which showed significantly enhanced uptake of HB/FA‐PEG‐PLA micelles in SKOV3 (FR+) compared to A2780 cancer cells (FR−). The enhanced uptake of HB/FA‐PEG‐PLA micelles to cancer cells resulted in a more effective post‐PDT killing of SKOV3 cells compared to plain micelles and free drugs. Binding and uptake of HB/FA‐PEG‐PLA micelles by SKOV3 cells were also observed in vivo after ip injection of folate‐targeted micelles in tumor‐bearing ascitic ovarian cancer animals. Drug levels in ascitic tumor tissues were increased 20‐fold (P < .001), which underscored the effect of a regional therapy approach with folate targeting. Furthermore, the HB‐loaded micelles were mainly distributed in kidney and liver (the main clearance organs) in biodistribution. These results showed that our newly developed PDT photosensitizer HB/FA‐PEG‐PLA micelles have a high drug‐loading capacity, good biocompatibility, controlled drug release, and enhanced targeting and antitumor effect, which is a potential approach to future targeting ovarian cancer therapy.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS).

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

INT-767 improves histopathological features in a diet-induced ob/ob mouse model of biopsy-confirmed non-alcoholic steatohepatitis.

AIMTo characterize the efficacy of the dual FXR/TGR5 receptor agonist INT-767 upon histological endpoints in a rodent model of diet-induced and biopsy-confirmed non-alcoholic steatohepatitis (NASH).METHODSThe effects of INT-767 on histological features of NASH were assessed in two studies using Lepob/ob (ob/ob) NASH mice fed the AMLN diet (high fat with trans-fat, cholesterol and fructose). In a proof-of-concept study, Lepob/ob (ob/ob) NASH mice were first dosed with INT-767 (3 or 10 mg/kg for 8 wk). A second ob/ob NASH study compared INT-767 (3 and 10 mg/kg) to obeticholic acid (OCA) (10 or 30 mg/kg; 16 wk). Primary histological endpoints included qualitative and quantitative assessments of NASH. Other metabolic and plasma endpoints were also assessed. A comparative assessment of INT-767 and OCA effects on drug distribution and hepatic gene expression was performed in C57Bl/6 mice on standard chow. C57Bl/6 mice were orally dosed with INT-767 or OCA (1-30 mg/kg) for 2 wk, and expression levels of candidate genes were assessed by RNA sequencing and tissue drug levels were measured by liquid chromatography tandem-mass spectrometry.RESULTSINT-767 dose-dependently (3 and 10 mg/kg, PO, QD, 8 wk) improved qualitative morphometric scores on steatohepatitis severity, inflammatory infiltrates and fibrosis stage. Quantitative morphometric analyses revealed that INT-767 reduced parenchymal collagen area, collagen fiber density, inflammation (assessed by Galectin-3 immunohistochemistry) and hepatocyte lipid droplet area following INT-767 treatment. In a comparative study (16 wk), the FXR agonists OCA (10 and 30 mg/kg) and INT-767 (3 and 10 mg/kg) both improved NASH histopathology, with INT-767 exerting greater therapeutic potency and efficacy than OCA. Mechanistic studies suggest that both drugs accumulate similarly within the liver and ileum, however, the effects of INT-767 may be driven by enhanced hepatic, but not ileal, FXR function.CONCLUSIONThese findings confirm the potential utility of FXR and dual FXR/TGR5 activation as disease intervention strategies in NASH.

Quantification of Thyromimetic Sobetirome Concentration in Biological Tissue Samples.

Thyroid hormone is a principal regulator of essential processes in vertebrate physiology and homeostasis. Synthetic derivatives of thyroid hormone, known as thyromimetics, display desirable therapeutic properties. Thoroughly understanding how thyromimetics distribute throughout the body is crucial for their development and this requires appropriate bioanalytical techniques to quantify drug levels in different tissues. Here, we describe a detailed protocol for the quantification of the thyromimetic sobetirome using liquid chromatography tandem-mass spectrometry (LC-MS/MS).

Development of a dietary formulation of the SHetA2 chemoprevention drug for mice

SummaryDevelopment of cancer chemoprevention compounds requires enhanced consideration for toxicity and route of administration because the target population is healthy. The small molecule drug, SHetA2 (NSC 726189), exhibited in vivo chemoprevention activity and lack of toxicity when administered by oral gavage. Our objective was to determine if a dietary formulation of SHetA2 could achieve effective tissue drug levels without toxicity. C57bl/6 J mice were monitored on modified American Institute of Nutrition (AIN)76A diet mixed with SHetA2 in a 3:1 ratio with Kolliphor HS15, a self-emulsifying drug delivery system (SEDDS) to deliver 37.5, 62.5, 125, 187 or 250 mg SHetA2/kg/day. Blood and tissues were evaluated after 1, 3 and 6 weeks. The 187 mg/kg/day dose was identified as optimal based on achievement of maximum blood and tissue drug levels in the effective micromolar range without evidence of toxicity. The 250 mg/kg/day group exhibited lower drug levels and the highest intestinal drug content suggesting that an upper limit of intestinal absorption had been surpassed. Only this highest dose resulted in liver and kidney function tests that were outside of the normal range, and significant reduction of cyclin D1 protein in normal cervical tissue. SHetA2 reduced cyclin D1 to greater extents in cancer compared to non-cancer cell cultures. Given this differential effect, optimal chemoprevention without toxicity would be expected to occur at doses that reduced cyclin D1 in neoplastic, but not in normal tissues. These findings support further development of SHetA2 as a chemoprevention agent and potential food additive.