Laura Pontiggia

A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme

Preclinical studies indicate autophagy inhibition with hydroxychloroquine (HCQ) can augment the efficacy of DNA-damaging therapy. The primary objective of this trial was to determine the maximum tolerated dose (MTD) and efficacy of HCQ in combination with radiation therapy (RT) and temozolomide (TMZ) for newly diagnosed glioblastoma (GB). A 3 + 3 phase I trial design followed by a noncomparative phase II study was conducted in GB patients after initial resection. Patients received HCQ (200 to 800 mg oral daily) with RT and concurrent and adjuvant TMZ. Quantitative electron microscopy and immunoblotting were used to assess changes in autophagic vacuoles (AVs) in peripheral blood mononuclear cells (PBMC). Population pharmacokinetic (PK) modeling enabled PK-pharmacodynamic correlations. Sixteen phase I subjects were evaluable for dose-limiting toxicities. At 800 mg HCQ/d, 3/3 subjects experienced Grade 3 and 4 neutropenia and thrombocytopenia, 1 with sepsis. HCQ 600 mg/d was found to be the MTD in this combination. The phase II cohort (n = 76) had a median survival of 15.6 mos with survival rates at 12, 18, and 24 mo of 70%, 36%, and 25%. PK analysis indicated dose-proportional exposure for HCQ. Significant therapy-associated increases in AV and LC3-II were observed in PBMC and correlated with higher HCQ exposure. These data establish that autophagy inhibition is achievable with HCQ, but dose-limiting toxicity prevented escalation to higher doses of HCQ. At HCQ 600 mg/d, autophagy inhibition was not consistently achieved in patients treated with this regimen, and no significant improvement in overall survival was observed. Therefore, a definitive test of the role of autophagy inhibition in the adjuvant setting for glioma patients awaits the development of lower-toxicity compounds that can achieve more consistent inhibition of autophagy than HCQ.

Combined MTOR and autophagy inhibition: Phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma

The combination of temsirolimus (TEM), an MTOR inhibitor, and hydroxychloroquine (HCQ), an autophagy inhibitor, augments cell death in preclinical models. This phase 1 dose-escalation study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with TEM in cancer patients. In the dose escalation portion, 27 patients with advanced solid malignancies were enrolled, followed by a cohort expansion at the top dose level in 12 patients with metastatic melanoma. The combination of HCQ and TEM was well tolerated, and grade 3 or 4 toxicity was limited to anorexia (7%), fatigue (7%), and nausea (7%). An MTD was not reached for HCQ, and the recommended phase II dose was HCQ 600 mg twice daily in combination with TEM 25 mg weekly. Other common grade 1 or 2 toxicities included fatigue, anorexia, nausea, stomatitis, rash, and weight loss. No responses were observed; however, 14/21 (67%) patients in the dose escalation and 14/19 (74%) patients with melanoma achieved stable disease. The median progression-free survival in 13 melanoma patients treated with HCQ 1200mg/d in combination with TEM was 3.5 mo. Novel 18-fluorodeoxyglucose positron emission tomography (FDG-PET) measurements predicted clinical outcome and provided further evidence that the addition of HCQ to TEM produced metabolic stress on tumors in patients that experienced clinical benefit. Pharmacodynamic evidence of autophagy inhibition was evident in serial PBMC and tumor biopsies only in patients treated with 1200 mg daily HCQ. This study indicates that TEM and HCQ is safe and tolerable, modulates autophagy in patients, and has significant antitumor activity. Further studies combining MTOR and autophagy inhibitors in cancer patients are warranted.

Combined autophagy and proteasome inhibition: A phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma

The efficacy of proteasome inhibition for myeloma is limited by therapeutic resistance, which may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation. Preclinical studies demonstrate that autophagy inhibition with hydroxychloroquine augments the antimyeloma efficacy of the proteasome inhibitor bortezomib. We conducted a phase I trial combining bortezomib and hydroxychloroquine for relapsed or refractory myeloma. We enrolled 25 patients, including 11 (44%) refractory to prior bortezomib. No protocol-defined dose-limiting toxicities occurred, and we identified a recommended phase 2 dose of hydroxychloroquine 600 mg twice daily with standard doses of bortezomib, at which we observed dose-related gastrointestinal toxicity and cytopenias. Of 22 patients evaluable for response, 3 (14%) had very good partial responses, 3 (14%) had minor responses, and 10 (45%) had a period of stable disease. Electron micrographs of bone marrow plasma cells collected at baseline, after a hydroxychloroquine run-in, and after combined therapy showed therapy-associated increases in autophagic vacuoles, consistent with the combined effects of increased trafficking of misfolded proteins to autophagic vacuoles and inhibition of their degradative capacity. Combined targeting of proteasomal and autophagic protein degradation using bortezomib and hydroxychloroquine is therefore feasible and a potentially useful strategy for improving outcomes in myeloma therapy.

Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma

Blocking autophagy with hydroxychloroquine (HCQ) augments cell death associated with alkylating chemotherapy in preclinical models. This phase I study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with dose-intense temozolomide (TMZ) in patients with advanced solid malignancies. Forty patients (73% metastatic melanoma) were treated with oral HCQ 200 to 1200 mg daily with dose-intense oral TMZ 150 mg/m2 daily for 7/14 d. This combination was well tolerated with no recurrent dose-limiting toxicities observed. An MTD was not reached for HCQ and the recommended phase II dose was HCQ 600 mg twice daily combined with dose-intense TMZ. Common toxicities included grade 2 fatigue (55%), anorexia (28%), nausea (48%), constipation (20%), and diarrhea (20%). Partial responses and stable disease were observed in 3/22 (14%) and 6/22 (27%) patients with metastatic melanoma. In the final dose cohort 2/6 patients with refractory BRAF wild-type melanoma had a near complete response, and prolonged stable disease, respectively. A significant accumulation in autophagic vacuoles (AV) in peripheral blood mononuclear cells was observed in response to combined therapy. Population pharmacokinetics (PK) modeling, individual PK simulations, and PK-pharmacodynamics (PD) analysis identified a threshold HCQ peak concentration that predicts therapy-associated AV accumulation. This study indicates that the combination of high-dose HCQ and dose-intense TMZ is safe and tolerable, and is associated with autophagy modulation in patients. Prolonged stable disease and responses suggest antitumor activity in melanoma patients, warranting further studies of this combination, or combinations of more potent autophagy inhibitors and chemotherapy in melanoma.

Anthelmintic screening of Sub-Saharan African plants used in traditional medicine

AIM OF STUDY This study screened for anthelmintic activity of plant species traditionally used in the treatment of intestinal parasites and their symptoms in Sub-Saharan Africa in an effort to confirm their local use and aid in the search for new compounds since resistance is a growing concern. MATERIALS AND METHODS Aqueous and organic extracts of 33 plant parts from 17 plant species traditionally used in the treatment of intestinal infections in Sub-Saharan Africa were evaluated for their anthelmintic activity. This activity was assessed using a standard motility assay against a levamisole resistant strain of the nematode Caenorhabditis elegans. RESULTS AND CONCLUSIONS Anthelmintic activity was confirmed in 12 plant species. Of these, eight showed strong evidence of activity (p<0.0001), one exhibited moderate evidence of activity (p<0.001), three demonstrated weak evidence of activity (p<0.05), and five plants showed no evidence of activity. The eight species with the strongest evidence of activity were Acacia polyacantha, Anogeissus leiocarpus, Bridelia micrantha, Cassia sieberiana, Combretum nigricans, Grewia bicolor, Strychnos spinosa and Ziziphus mucronata. In only two cases, Anogeissus leiocarpus and Cassia sieberiana, anthelmintic activity has been previously confirmed. The activity demonstrated against the levamisole resistant strain of Caenorhabditis elegans and the presence of molecules in these plants known or suspected of having a broad spectrum of activity provide support for further study of these plants and their compounds as possible treatments for parasitic worm infections.

Elucidation of thrifty features in adult rats exposed to protein restriction during gestation and lactation

Since the introduction of the thrifty phenotype hypothesis, the potential traits of thrift have been described in increasingly broad terms but biochemical and behavioral evidence of thrift has not been well demonstrated. The objective of our studies was to use a rodent model to identify features of thrift programmed by early life protein restriction. Robust programming of thrifty features requires a thrifty nutritional environment during the entire window of developmental plasticity. Therefore, pregnant rats were exposed to a low protein diet throughout the window of developmental plasticity spanning the period of gestation and lactation and its effects on energy acquisition, storage and expenditure in the adult offspring were examined. Maternal protein restriction reduced birth weight and produced long term reductions in body and organ weights in the offspring. Low protein offspring demonstrated an increased drive to seek food as evidenced by hyperphagia that was mediated by changes in plasma leptin and ghrelin levels. Hyperphagia was accompanied by increased efficiency in converting caloric intake into body mass. The higher feed efficiency was mediated by greater insulin sensitivity. Energy expenditure of low protein offspring in locomotion was not affected either in the light or dark phase. However, low protein offspring exhibited higher resting and basal metabolic rates as evidenced by higher core body temperature in the fed and fasted states. The increased thermogenesis was not mediated by thyroid hormones but by an increased sympathetic nervous system drive as reflected by a lower areal bone mineral density and bone mineral content and lower plasma adiponectin and triglyceride levels. Elevated thermogenesis in the low protein offspring possibly offsets the effects of hyperphagia, minimizes their chances of weight gain, and improves survivability. This constellation of metabolic features in the low protein offspring will maximize survival potential in a post natal environment of nutritional scarcity and constitute a thrifty phenotype.

Comparison of motility, recovery, and methyl-thiazolyl-tetrazolium reduction assays for use in screening plant products for anthelmintic activity

The primary objective of these experiments was to compare the effectiveness of motility, recovery, and methyl-thiazolyl-tetrazolium (MTT) reduction assays for determining anthelmintic activity of plant extracts and purified compounds from these extracts. Caenorhabditis elegans was used as the test organism. High-performance liquid chromatography (HPLC) grade water and M9 medium were used as the solvents. Copper, a common metal pollutant, and the anthelmintic drug levamisole were used as reference compounds. Extracts from the West African plant Anogeissus leiocarpus, which is used to treat worm infections, as well as two active compounds found in this plant, gallic and gentisic acids, were included in this comparison. MTT assay results for viability of worms were significantly lower (p < 0.01) than motility and recovery assay results. However, both gallic acid and the plant extract, in the absence of worms, caused reduction of MTT. Worm survival for levamisole using M9 medium was significantly (p < 0.01) higher than for HPLC grade water for all three methods. On the other hand, gallic acid showed significant (p < 0.05) activity in M9 medium but no activity in HPLC grade water, whereas gentisic acid was effective in HPLC grade water but had no activity in M9 medium. Activity of the A. leiocarpus extract also varied with solvent. In conclusion, plant extracts can be screened using motility assays that include both HPLC grade water and M9 salts.

Aqueous and vitreous penetration of linezolid and levofloxacin after oral administration.

PURPOSE To evaluate the time course of drug concentrations achieved in aqueous (AQ), vitreous (V), and serum (S) compartments after oral administration of linezolid and levofloxacin. DESIGN Randomized, clinical trial. METHODS SETTINGS Clinical practice. PATIENT POPULATION Sixteen patients (16 eyes) undergoing vitrectomy who had not had a prior pars plana vitrectomy in the study eye were randomly assigned to one of 4 groups. INTERVENTION AQ, V, and S samples were obtained from all subjects after single concomitant doses of linezolid 600 mg and levofloxacin 750 mg between 1 and 12 h before the procedure: group A = 1-3 h; group B = 3-6 h; group C = 6-9 h; group D = 9-12 h. MAIN OUTCOME MEASURES AQ, V, and S concentrations of linezolid and levofloxacin. RESULTS Overall mean concentrations ± standard deviation (μg/mL) achieved by linezolid in AQ, V, and S compartments were 3.32 ± 2.06, 2.98 ± 1.87, and 7.91 ± 3.94, respectively. Overall mean concentrations ±standard deviation (μg/mL) achieved by levofloxacin in AQ, V, and S compartments were 2.19 ± 1.92, 1.95 ± 1.27, and 7.38 ± 3.47, respectively. CONCLUSIONS Single concomitant doses of linezolid and levofloxacin achieved AQ and V concentrations above the minimum inhibitory concentration for 90% of common ocular gram-positive and gram-negative pathogens up to 12 h after administration. The combination of linezolid and levofloxacin represents a viable option for the prophylaxis and management of endophthalmitis.

Maternal protein restriction during pregnancy and lactation alters central leptin signalling, increases food intake, and decreases bone mass in 1 year old rat offspring.

The effects of perinatal nutrition on offspring physiology have mostly been examined in young adult animals. Aging constitutes a risk factor for the progressive loss of metabolic flexibility and development of disease. Few studies have examined whether the phenotype programmed by perinatal nutrition persists in aging offspring. Persistence of detrimental phenotypes and their accumulative metabolic effects are important for disease causality. This study determined the effects of maternal protein restriction during pregnancy and lactation on food consumption, central leptin sensitivity, bone health, and susceptibility to high fat diet‐induced adiposity in 1‐year‐old male offspring. Sprague‐Dawley rats received either a control or a protein restricted diet throughout pregnancy and lactation and pups were weaned onto laboratory chow. One‐year‐old low protein (LP) offspring exhibited hyperphagia. The inability of an intraperitoneal (i.p.) leptin injection to reduce food intake indicated that the hyperphagia was mediated by decreased central leptin sensitivity. Hyperphagia was accompanied by lower body weight suggesting increased energy expenditure in LP offspring. Bone density and bone mineral content that are negatively regulated by leptin acting via the sympathetic nervous system (SNS), were decreased in LP offspring. LP offspring did not exhibit increased susceptibility to high fat diet induced metabolic effects or adiposity. The results presented here indicate that the programming effects of perinatal protein restriction are mediated by specific decreases in central leptin signalling to pathways involved in the regulation of food intake along with possible enhancement of different CNS leptin signalling pathways acting via the SNS to regulate bone mass and energy expenditure.

Decreased liver triglyceride content in adult rats exposed to protein restriction during gestation and lactation: Role of hepatic triglyceride utilization

We have previously demonstrated that protein restriction throughout gestation and lactation reduces liver triglyceride content in adult rat offspring. However, the mechanisms mediating the decrease in liver triglyceride content are not understood. The aim of the current study was to use a new group of pregnant animals and their offspring and determine the contribution of increased triglyceride utilization via the hepatic fatty‐acid oxidation and triglyceride secretory pathways to the reduction in liver triglyceride content. Pregnant Sprague–Dawley rats received either a control or a low protein diet throughout pregnancy and lactation. Pups were weaned onto laboratory chow on day 28 and killed on day 65. Liver triglyceride content was reduced in male, but not female, low‐protein offspring, both in the fed and fasted states. The reduction was accompanied by a trend towards higher liver carnitine palmitoyltransferase‐1a activity, suggesting increased fatty‐acid transport into the mitochondrial matrix. However, medium‐chain acyl coenzyme A dehydrogenase activity within the mitochondrial matrix, expression of nuclear peroxisome proliferator activated receptor‐α, and plasma levels of β‐hydroxybutyrate were similar between low protein and control offspring, indicating a lack of change in fatty‐acid oxidation. Hepatic triglyceride secretion, assessed by blocking peripheral triglyceride utilization and measuring serum triglyceride accumulation rate, and the activity of microsomal transfer protein, were similar between low protein and control offspring. Because enhanced triglyceride utilization is not a significant contributor, the decrease in liver triglyceride content in male low‐protein offspring is likely due to alterations in liver fatty‐acid transport or triglyceride biosynthesis.