Phase 1 trial of ADI-PEG 20 and liposomal doxorubicin in patients with metastatic solid tumors.

BACKGROUND: Arginine depletion interferes with pyrimidine metabolism and DNA damage repair pathways. Preclinical data demonstrated that depletion of arginine by PEGylated arginine deiminase (ADI-PEG 20) enhanced liposomal doxorubicin (PLD) cytotoxicity in cancer cells with argininosuccinate synthase 1 (ASS1) deficiency. The objective of this study was to assess safety and tolerability of ADI-PEG 20 and PLD in patients with metastatic solid tumors. METHODS: Patients with advanced ASS1-deficient solid tumors were enrolled in this phase 1 trial of ADI-PEG 20 and PLD following a 3 + 3 design. Eligible patients were given intravenous PLD biweekly and intramuscular (IM) ADI-PEG 20 weekly. Toxicity and efficacy were evaluated according to the Common Terminology Criteria for Adverse Events (version 4.0) and Response Evaluation Criteria in Solid Tumors (version 1.1), respectively. RESULTS: Of 15 enrolled patients, 9 had metastatic HER2-negative breast carcinoma. We observed no dose-limiting toxicities or treatment-related deaths. One patient safely received 880 mg/m2 PLD in this study and 240 mg/m2 doxorubicin previously. Treatment led to stable disease in 9 patients and was associated with a median progression-free survival time of 3.95 months in 15 patients. Throughout the duration of treatment, decreased arginine and increased citrulline levels in peripheral blood remained significant in a majority of patients. We detected no induction of anti-ADI-PEG 20 antibodies by week 8 in one third of patients. CONCLUSION: Concurrent IM injection of ADI-PEG 20 at 36 mg/m2 weekly and intravenous infusion of PLD at 20 mg/m2 biweekly had an acceptable safety profile in patients with advanced ASS1-deficient solid tumors. Further evaluation of this combination is under discussion.

Meta-analysis identifies the effect of dietary multi-enzyme supplementation on gut health of pigs.

Gut health though is not well defined the role of gastrointestinal tract is vital if an animal must perform well. Apart from digestion, secretion, and absorption gut is harbored with consortium of microbiota which plays a key role in one's health. Enzymes, one of the alternatives for antibiotics with beneficial effects on digestion and consistency of food and its effect on gut health. The effect of enzyme supplementation on gut health is not well established and the objective of this meta-analysis is to investigate if the enzyme supplement has influence on gut. This meta-analysis includes 1221 experiments which has single enzyme studies and or studies with multiple enzyme complexes but not challenged. The ratio of Lactobacillus and E. coli is related to ADFI which showed comparatively lower negative correlation coefficient, with - 0.052 and - 0.035, respectively, whose I2 values are below 25%, showing that these studies show a significantly lower level of heterogeneity. Correlation between villus height, crypt depth, their ratio and fatty acid is also assessed, and it showed that when the animal is supplemented with two enzyme complexes resulted in positive gut health rather than the single or more than two enzymes.

Phase I study of ADI-PEG20 plus low-dose cytarabine for the treatment of acute myeloid leukemia.

Most acute myeloid leukemia (AML) cells are argininosuccinate synthetase-deficient. Pegylated arginine deiminase (ADI-PEG20) monotherapy depletes circulating arginine, thereby selectively inducing tumor cell death. ADI-PEG20 was shown to induce complete responses in ~10% of relapsed/refractory or poor-risk AML patients. We conducted a phase I, dose-escalation study combining ADI-PEG20 and low-dose cytarabine (LDC) in AML patients. Patients received 20 mg LDC subcutaneously twice daily for 10 days every 28 days and ADI-PEG20 at 18 or 36 mg/m2 (dose levels 1 and 2) intramuscularly weekly. An expansion cohort for the maximal tolerated dose of ADI-PEG20 was planned to further estimate the toxicity and preliminary response of this regimen. The primary endpoints were safety and tolerability. The secondary endpoints were time on treatment, overall survival (OS), overall response rate (ORR), and biomarkers (pharmacodynamics and immunogenicity detection). Twenty-three patients were included in the study, and seventeen patients were in the expansion cohort (dose level 2). No patients developed dose-limiting toxicities. The most common grade III/IV toxicities were thrombocytopenia (61%), anemia (52%), and neutropenia (30%). One had an allergic reaction to ADI-PEG20. The ORR in 18 evaluable patients was 44.4%, with a median OS of 8.0 (4.5-not reached) months. In seven treatment-naïve patients, the ORR was 71.4% and the complete remission rate was 57.1%. The ADI-PEG20 and LDC combination was well-tolerated and resulted in an encouraging ORR. Further combination studies are warranted. (This trial was registered in ClinicalTrials.gov as a Ph1 Study of ADI-PEG20 Plus Low-Dose Cytarabine in Older Patients With AML, NCT02875093).

Phase 1 trial of ADI-PEG20 plus cisplatin in patients with pretreated metastatic melanoma or other advanced solid malignancies.

BACKGROUND: Arginine depletion interferes with pyrimidine metabolism and DNA damage-repair pathways, and pairing arginine deiminase pegylated with 20,000-molecular-weight polyethylene glycol (ADI-PEG20) with platinum enhances cytotoxicity in vitro and in vivo in arginine auxotrophs. METHODS: This single-centre, Phase 1 trial was conducted using a 3 + 3 dose escalation designed to assess safety, tolerability and determine the recommended Phase 2 dose (RP2D) of ADI-PEG20. RESULTS: We enrolled 99 patients with metastatic argininosuccinate synthetase 1 (ASS1) deficient malignancies. We observed no dose-limiting toxic effects or treatment-related mortality. Three percent of patients discontinued treatment because of toxicity. After treatment, 5% (5/99) of patients had partial responses, and 41% had stable disease. The median progression-free and overall survival durations were 3.62 and 8.06 months, respectively. Substantial arginine depletion and citrulline escalation persisted in most patients through weeks 24 and 8, respectively. Tumour responses were associated with anti-ADI-PEG20 antibody levels at weeks 8 and 16 (p = 0.031 and p = 0.0357, respectively). CONCLUSION: Concurrently administered ADI-PEG20 and cisplatin had an acceptable safety profile and had shown antitumour activity against metastatic ASS1-deficient solid tumours. Further evaluation of this treatment combination is warranted.

Metabolism of Zearalenone in the Rumen of Dairy Cows with and without Application of a Zearalenone-Degrading Enzyme.

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feed and is well known for its estrogenic effects in animals. Cattle are considered less sensitive to ZEN than pigs. However, ZEN has previously been shown to be converted to the highly estrogenic metabolite α-zearalenol (α-ZEL) in rumen fluid in vitro. Here, we investigate the metabolism of ZEN in the reticulorumen of dairy cows. To this end, rumen-fistulated non-lactating Holstein Friesian cows (n = 4) received a one-time oral dose of ZEN (5 mg ZEN in 500 g concentrate feed) and the concentrations of ZEN and ZEN metabolites were measured in free rumen liquid from three reticulorumen locations (reticulum, ventral sac and dorsal mat layer) during a 34-h period. In all three locations, α-ZEL was the predominant ZEN metabolite and ß-zearalenol (ß-ZEL) was detected in lower concentrations. ZEN, α-ZEL and ß-ZEL were eliminated from the ventral sac and reticulum within 34 h, yet low concentrations of ZEN and α-ZEL were still detected in the dorsal mat 34 h after ZEN administration. In a second step, we investigated the efficacy of the enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) to degrade ZEN to the non-estrogenic metabolite hydrolyzed zearalenone (HZEN) in the reticulorumen in vitro and in vivo. ZenA showed a high ZEN-degrading activity in rumen fluid in vitro. When ZenA was added to ZEN-contaminated concentrate fed to rumen-fistulated cows (n = 4), concentrations of ZEN, α-ZEL and ß-ZEL were significantly reduced in all three reticulorumen compartments compared to administration of ZEN-contaminated concentrate without ZenA. Upon ZenA administration, degradation products HZEN and decarboxylated HZEN were detected in the reticulorumen. In conclusion, endogenous metabolization of ZEN in the reticulorumen increases its estrogenic potency due to the formation of α-ZEL. Our results suggest that application of zearalenone hydrolase ZenA as a feed additive may be a promising strategy to counteract estrogenic effects of ZEN in cattle.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.

Green Chemistry Extractions of Carotenoids from Daucus carota L.-Supercritical Carbon Dioxide and Enzyme-Assisted Methods.

Multiple reviews have been published on various aspects of carotenoid extraction. Nevertheless, none of them focused on the discussion of recent green chemistry extraction protocols, especially for the carotenoids extraction from Daucus carota L. This group of bioactive compounds has been chosen for this review since most of the scientific papers proved their antioxidant properties relevant for inflammation, stress-related disorders, cancer, or neurological and neurodegenerative diseases, such as stroke and Alzheimer's Disease. Besides, carrots constitute one of the most popular sources of carotenoids. In the presented review emphasis has been placed on the supercritical carbon dioxide and enzyme-assisted extraction techniques for the relevant tetraterpenoids. The detailed descriptions of these methods, as well as practical examples, are provided. In addition, the pros and cons of each method and comparison with the standard solvent extraction have been discussed.

Efficacy, safety and immunogenicity of a pneumococcal protein-based vaccine co-administered with 13-valent pneumococcal conjugate vaccine against acute otitis media in young children: A phase IIb randomized study.

BACKGROUND: Native American populations experience a substantial burden of pneumococcal disease despite use of highly effective pneumococcal conjugate vaccines (PCVs). Protein-based pneumococcal vaccines may extend protection beyond the serotype-specific protection elicited by PCVs. METHODS: In this phase IIb, double-blind, controlled trial, 6-12 weeks-old Native American infants randomized 1:1, received either a protein-based pneumococcal vaccine (dPly/PhtD) containing pneumolysin toxoid (dPly, 10 µg) and pneumococcal histidine triad protein D (PhtD, 10 µg) or placebo, administered along with 13-valent PCV (PCV13) at ages 2, 4, 6 and 12-15 months. Other pediatric vaccines were given per the routine immunization schedule. We assessed vaccine efficacy (VE) against acute otitis media (AOM) and acute lower respiratory tract infection (ALRI) endpoints. Immunogenicity, reactogenicity and unsolicited adverse events were assessed in a sub-cohort and serious adverse events were assessed in all children. RESULTS: 1803 infants were randomized (900 dPly/PhtD; 903 Control). VE against all episodes of American Academy of Pediatrics (AAP)-defined AOM was 3.8% (95% confidence interval: -11.4, 16.9). Point estimates of VE against other AOM outcomes ranged between 2.9% (-9.5, 14.0) and 5.2% (-8.0, 16.8). Point estimates of VE against ALRI outcomes ranged between -4.4% (-39.2, 21.8) and 2.0% (-18.3, 18.8). Point estimates of VE tended to be higher against first than all episodes but the confidence intervals included zero. dPly/PhtD vaccine was immunogenic and had an acceptable reactogenicity and safety profile after primary and booster vaccination in Native American infants. CONCLUSIONS: The dPly/PhtD vaccine was immunogenic and well tolerated, however, incremental efficacy in preventing AAP-AOM over PCV13 was not demonstrated. CLINICAL TRIALS REGISTRATION: NCT01545375 (www.clinicaltrials.gov).

A Phase I Study of Pegylated Arginine Deiminase (Pegargiminase), Cisplatin, and Pemetrexed in Argininosuccinate Synthetase 1-Deficient Recurrent High-grade Glioma.

PURPOSE: Patients with recurrent high-grade gliomas (HGG) are usually managed with alkylating chemotherapy ± bevacizumab. However, prognosis remains very poor. Preclinically, we showed that HGGs are a target for arginine depletion with pegargiminase (ADI-PEG20) due to epimutations of argininosuccinate synthetase (ASS1) and/or argininosuccinate lyase (ASL). Moreover, ADI-PEG20 disrupts pyrimidine pools in ASS1-deficient HGGs, thereby impacting sensitivity to the antifolate, pemetrexed. PATIENTS AND METHODS: We expanded a phase I trial of ADI-PEG20 with pemetrexed and cisplatin (ADIPEMCIS) to patients with ASS1-deficient recurrent HGGs (NCT02029690). Patients were enrolled (01/16-06/17) to receive weekly ADI-PEG20 36 mg/m2 intramuscularly plus pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 intravenously once every 3 weeks for up to 6 cycles. Patients with disease control were allowed ADI-PEG20 maintenance. The primary endpoints were safety, tolerability, and preliminary estimates of efficacy. RESULTS: Ten ASS1-deficient heavily pretreated patients were treated with ADIPEMCIS therapy. Treatment was well tolerated with the majority of adverse events being Common Terminology Criteria for Adverse Events v4.03 grade 1-2. The best overall response was stable disease in 8 patients (80%). Plasma arginine was suppressed significantly below baseline with a reciprocal increase in citrulline during the sampling period. The anti-ADI-PEG20 antibody titer rose during the first 4 weeks of treatment before reaching a plateau. Median progression-free survival (PFS) was 5.2 months (95% confidence interval (CI), 2.5-20.8) and overall survival was 6.3 months (95% CI, 1.8-9.7). CONCLUSIONS: In this recurrent HGG study, ADIPEMCIS was well tolerated and compares favorably to historical controls. Additional trials of ADI-PEG20 in HGG are planned.

Endoluminal Atherosclerotic Plaque Debulking Using Enzymatic and Ultrasonic Energy.

BACKGROUND: Current procedures to treat severe atherosclerosis are traumatic to the arterial wall and often result in restenosis due to neointimal hyperplasia. We developed a novel therapy using a specially designed double occlusion balloon catheter, ultrasonic wire, and enzymatic digestion solution to atraumatically debulk atherosclerotic plaques. MATERIALS AND METHODS: A combination of different enzymes, chemicals, and treatment conditions were evaluated for its effect at reducing atherosclerotic plaque harvested from human carotid artery endarterectomies ex vivo. The optimized digestion solution was examined in harvested intact human superficial femoral arteries in situ. A conventional Yorkshire/Landrace and a genetically modified Yucatan minipig homozygous for a nonfunctional LDLR mutation were used to evaluate the endovascular therapy in nonatherosclerotic and atherosclerotic environments in vivo. RESULTS: Ex vivo, the technology successfully digested human carotid artery plaques by 75%. In situ, the therapy successfully reduced plaque area in harvested superficial femoral arteries by 46%. In vivo, the endovascular therapy was technically feasible and demonstrated initial safety with no thrombosis, dissection, or aneurysmal dilatation in a nonatherosclerotic porcine model. In an atherosclerotic porcine model, the therapy demonstrated initial efficacy by successfully reducing atherosclerotic plaque while preserving the arterial wall with an intact internal elastic lamina. CONCLUSIONS: Using human plaque, human artery, and a normal and atherosclerotic pig model, we demonstrated that delivery of our therapy to the vasculature is technically feasible, appears safe, and shows initial efficacy. Our percutaneous plaque debulking method is a unique and promising therapy for the treatment of atherosclerosis and warrants further study.

A phase 1 study of ADI-PEG 20 and modified FOLFOX6 in patients with advanced hepatocellular carcinoma and other gastrointestinal malignancies.

PURPOSE: Arginine depletion interferes with pyrimidine metabolism as well as DNA damage repair pathways. Preclinical data indicates that pairing pegylated arginine deiminase (ADI-PEG 20) with fluoropyrimidines or platinum enhances cytotoxicity in vitro and in vivo in arginine auxotrophs. METHODS: This is a single-center, open-label, phase 1 trial of ADI-PEG 20 and modified FOLFOX6 (mFOLFOX6) in treatment-refractory hepatocellular carcinoma (HCC) and other advanced gastrointestinal tumors. A 3 + 3 dose escalation design was employed to assess safety, tolerability, and determine the recommended phase 2 dose (RP2D) of ADI-PEG 20. A RP2D expansion cohort for patients with HCC was employed to define the objective response rate (ORR). Secondary objectives were to estimate progression-free survival (PFS), overall survival (OS), and to explore pharmacodynamics and immunogenicity. Eligible patients were treated with mFOLFOX6 intravenously biweekly at standard doses and ADI-PEG-20 intramuscularly weekly at 18 (Cohort 1) or 36 mg/m2 (Cohort 2 and RP2D expansion). RESULTS: Twenty-seven patients enrolled-23 with advanced HCC and 4 with other gastrointestinal tumors. No dose-limiting toxicities were observed in cohort 1 or 2. The RP2D for ADI-PEG 20 was 36 mg/m2 weekly with mFOLFOX6. The most common any grade adverse events (AEs) were thrombocytopenia, neutropenia, leukopenia, anemia, and fatigue. Among the 23 HCC patients, the most frequent treatment-related Grade ≥ 3 AEs were neutropenia (47.8%), thrombocytopenia (34.7%), leukopenia (21.7%), anemia (21.7%), and lymphopenia (17.4%). The ORR for this group was 21% (95% CI 7.5-43.7). Median PFS and OS were 7.3 and 14.5 months, respectively. Arginine levels were depleted with therapy despite the emergence of low levels of anti-ADI-PEG 20 antibodies. Arginine depletion at 4 and 8 weeks and archival tumoral argininosuccinate synthetase-1 levels did not correlate with response. CONCLUSIONS: Concurrent mFOLFOX6 plus ADI-PEG-20 intramuscularly at 36 mg/m2 weekly shows an acceptable safety profile and favorable efficacy compared to historic controls. Further evaluation of this combination is warranted in advanced HCC patients.

A phase 1/1B trial of ADI-PEG 20 plus nab-paclitaxel and gemcitabine in patients with advanced pancreatic adenocarcinoma.

BACKGROUND: ADI-PEG 20 is a pegylated form of the arginine-depleting enzyme arginine deiminase. Normal cells synthesize arginine with the enzyme argininosuccinate synthetase (ASS1); ADI-PEG 20 selectively targets malignant cells, which lack ASS1. METHODS: A single-arm, nonrandomized, open-label, phase 1/1B, standard 3 + 3 dose escalation with an expansion cohort of 9 patients at the recommended phase 2 dose (RP2D) was conducted. Patients who had metastatic pancreatic cancer, up to 1 line of prior treatment (the dose-escalation cohort) or no prior treatment (the expansion cohort), and an Eastern Cooperative Oncology Group performance status of 0 to 1 were included. Patients received both gemcitabine (1000 mg/m2 ) and nab-paclitaxel (125 mg/m2 ) for 3 of 4 weeks and intramuscular ADI-PEG 20 at 18 mg/m2 weekly (cohort 1) or at 36 mg/m2 weekly (cohort 2 and the expansion cohort).The primary endpoint was to determine the maximum tolerated dose and RP2D of ADI-PEG 20 in combination with nab-paclitaxel and gemcitabine. RESULTS: Eighteen patients were enrolled. No dose-limiting toxicities (DLTs) were observed in cohort 1; cohort 2 was expanded to 6 patients because of 1 DLT occurrence (a grade 3 elevation in bilirubin, aspartate aminotransferase, and alanine aminotransferase). The most frequent adverse events (AEs) of any grade were neutropenia, thrombocytopenia, leukopenia, anemia, peripheral neuropathy, and fatigue; all 18 patients experienced grade 3/4 AEs. The most frequent grade 3/4 toxicities, regardless of the relation with any drugs, included neutropenia (12 patients or 67%), leukopenia (10 patients or 56%), anemia (8 patients or 44%), and lymphopenia (6 patients or 33%). The RP2D for ADI-PEG 20 was 36 mg/m2 weekly in combination with standard-dose gemcitabine and nab-paclitaxel. The overall response rate among patients treated at the RP2D in the first-line setting was 45.5% (5 of 11).The median progression-free survival time for these patients treated at the RP2D was 6.1 months (95% confidence interval, 5.3-11.2 months), and the median overall survival time was 11.3 months (95% confidence interval, 6.7 months to not reached). CONCLUSIONS: ADI-PEG 20 was well tolerated in combination with gemcitabine and nab-paclitaxel. Activity was observed in previously treated and untreated patients with advanced pancreatic cancer and in patients with ASS1-deficient and -proficient tumors. Cancer 2017;123:4556-4565. © 2017 American Cancer Society.

Phase 1 Dose-Escalation Study of Pegylated Arginine Deiminase, Cisplatin, and Pemetrexed in Patients With Argininosuccinate Synthetase 1-Deficient Thoracic Cancers.

Purpose Pegylated arginine deiminase (ADI-PEG 20) depletes essential amino acid levels in argininosuccinate synthetase 1 (ASS1) -negative tumors by converting arginine to citrulline and ammonia. The main aim of this study was to determine the recommended dose, safety, and tolerability of ADI-PEG 20, cisplatin, and pemetrexed in patients with ASS1-deficient malignant pleural mesothelioma (MPM) or non-small-cell lung cancer (NSCLC). Patients and Methods Using a 3 + 3 + 3 dose-escalation study, nine chemotherapy-naïve patients (five MPM, four NSCLC) received weekly ADI-PEG 20 doses of 18 mg/m2, 27 mg/m2, or 36 mg/m2, together with pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 which were given every three weeks (maximum of six cycles). Patients achieving stable disease or better could continue ADI-PEG 20 monotherapy until disease progression or withdrawal. Adverse events were assessed by Common Terminology Criteria for Adverse Events version 4.03, and pharmacodynamics and immunogenicity were also evaluated. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 for NSCLC and by modified RECIST criteria for MPM. Results No dose-limiting toxicities were reported; nine of 38 reported adverse events (all grade 1 or 2) were related to ADI-PEG 20. Circulating arginine concentrations declined rapidly, and citrulline levels increased; both changes persisted at 18 weeks. Partial responses were observed in seven of nine patients (78%), including three with either sarcomatoid or biphasic MPM. Conclusion Target engagement with depletion of arginine was maintained throughout treatment with no dose-limiting toxicities. In this biomarker-selected group of patients with ASS1-deficient cancers, clinical activity was observed in patients with poor-prognosis tumors. Therefore, we recommend a dose for future studies of weekly ADI-PEG 20 36 mg/m2 plus three-weekly cisplatin 75 mg/m2 and pemetrexed 500 mg/m2.

Protective effect of antigen excess immune complex in guinea pigs infected with Mycobacterium tuberculosis.

Background & objectives: : Immune complexes (ICs) play a crucial role which can either be beneficial or pathological to the host. Involvement of circulating immune complexes (CICs) has been shown in tuberculosis (TB) cases (adults and neonates form), but its immunomodulatory effect has not been studied in vivo. Hence, this study was carried out to understand and explore the prognostic therapeutic potential of CICs on the host immune system in guinea pigs animal TB model. Methods: In this study, the guinea pigs (group I) were immunized with in vitro synthesized antigen excess IC (AgX-IC), group II with antibody excess IC (AbX-IC) and group III with phosphate-buffered saline. All these animals were sensitized with Mycobacterium tuberculosis H37Rv before immunization and subsequently infected with M. tuberculosis H37Rv strain post-immunization with IC. Results: Mortality was observed in animals belonging of groups II and III, while all animals in group I survived. A steady increase in the body weight of animals immunized with AgX-IC was observed when compared to the other groups. The infection load in the spleen and lungs was less in animals from group I when compared to the other groups. The CICs were found to be in higher concentration in serum of IC-immunized guinea pigs when compared to ICs non-immunized animals. Interpretation & conclusions: Based on our findings, it can be speculated that the ICs may have a protective immunomodulatory role pertaining to disease progression and development of pathology. As a new perspective, with further insight into the underlying mechanism of action and correlation with clinical data, ICs may also be used as a potential tool for assessing the immune status of the infected individuals, especially the close contacts of TB patients.

Arginine Deprivation With Pegylated Arginine Deiminase in Patients With Argininosuccinate Synthetase 1-Deficient Malignant Pleural Mesothelioma: A Randomized Clinical Trial.

IMPORTANCE: Preclinical studies show that arginine deprivation is synthetically lethal in argininosuccinate synthetase 1 (ASS1)-negative cancers, including mesothelioma. The role of the arginine-lowering agent pegylated arginine deiminase (ADI-PEG20) has not been evaluated in a randomized and biomarker-driven study among patients with cancer. OBJECTIVE: To assess the clinical impact of arginine depletion in patients with ASS1-deficient malignant pleural mesothelioma. DESIGN, SETTING, AND PARTICIPANTS: A multicenter phase 2 randomized clinical trial, the Arginine Deiminase and Mesothelioma (ADAM) study, was conducted between March 2, 2011, and May 21, 2013, at 8 academic cancer centers. Immunohistochemical screening of 201 patients (2011-2013) identified 68 with advanced ASS1-deficient malignant pleural mesothelioma. INTERVENTIONS: Randomization 2:1 to arginine deprivation (ADI-PEG20, 36.8 mg/m2, weekly intramuscular) plus best supportive care (BSC) or BSC alone. MAIN OUTCOMES AND MEASURES: The primary end point was progression-free survival (PFS) assessed by modified Response Evaluation Criteria in Solid Tumors (RECIST) (target hazard ratio, 0.60). Secondary end points were overall survival (OS), tumor response rate, safety, and quality of life, analyzed by intention to treat. We measured plasma arginine and citrulline levels, anti-ADI-PEG20 antibody titer, ASS1 methylation status, and metabolic response by 18F-fluorodeoxyglucose positron-emission tomography. RESULTS: Median (range) follow-up in 68 adults (median [range] age, 66 [48-83] years; 19% female) was 38 (2.5-39) months. The PFS hazard ratio was 0.56 (95% CI, 0.33-0.96), with a median of 3.2 months in the ADI-PEG20 group vs 2.0 months in the BSC group (P = .03) (absolute risk, 18% vs 0% at 6 months). Best response at 4 months (modified RECIST) was stable disease: 12 of 23 (52%) in the ADI-PEG20 group vs 2 of 9 (22%) in the BSC group (P = .23). The OS curves crossed, so life expectancy was used: 15.7 months in the ADI-PEG20 group vs 12.1 months in the BSC group (difference of 3.6 [95% CI, -1.0 to 8.1] months; P = .13). The incidence of symptomatic adverse events of grade at least 3 was 11 of 44 (25%) in the ADI-PEG20 group vs 4 of 24 (17%) in the BSC group (P = .43), the most common being immune related, nonfebrile neutropenia, gastrointestinal events, and fatigue. Differential ASS1 gene-body methylation correlated with ASS1 immunohistochemistry, and longer arginine deprivation correlated with improved PFS. CONCLUSIONS AND RELEVANCE: In this trial, arginine deprivation with ADI-PEG20 improved PFS in patients with ASS1-deficient mesothelioma. Targeting arginine is safe and warrants further clinical investigation in arginine-dependent cancers. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01279967.

Slow, continuous enzyme replacement via spinal CSF in dogs with the paediatric-onset neurodegenerative disease, MPS IIIA.

Intra-cerebrospinal fluid (CSF) injection of recombinant human lysosomal enzyme is a potential treatment strategy for several neurodegenerative lysosomal storage disorders including Sanfilippo syndrome (Mucopolysaccharidosis type IIIA; MPS IIIA). Here we have utilised the MPS IIIA Huntaway dog model to compare the effectiveness of the repeated intermittent bolus injection strategy being used in the trials with an alternate approach; slow, continual infusion of replacement enzyme (recombinant human sulphamidase; rhSGSH) into the spinal CSF using a SynchroMed II® pump attached to a spinal infusion cannula. The ability of each enzyme delivery strategy to ameliorate lesions in MPS IIIA brain was determined in animals treated from ∼three- to six-months of age. Controls received buffer or no treatment. Significant reductions in heparan sulphate (primary substrate) were observed in brain samples from dogs treated via either cisternal or lumbar spinal CSF bolus injection methods and also in slow intra-spinal CSF infusion-treated dogs. The extent of the reduction differed regionally. Pump-delivered rhSGSH was less effective in reducing secondary substrate (GM3 ganglioside) in deeper aspects of cerebral cortex, and although near-amelioration of microglial activation was seen in superficial (but not deep) layers of cerebral cortex in both bolus enzyme-treated groups, pump-infusion of rhSGSH had little impact on microgliosis. While continual low-dose infusion of rhSGSH into MPS IIIA dog CSF reduces disease-based lesions in brain, it was not as efficacious as repeated cisternal or spinal CSF bolus infusion of rhSGSH over the time-frame of these experiments.

Brain Targeting in MPS-IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a childhood metabolic neuropathology caused by the inherited deficiency of the lysosomal enzyme sulfamidase and is characterized by the accumulation of undegraded glycosaminoglycans in the lysosomes of cells and tissues of affected patients. MPS-IIIA represents one of the most common forms of lysosomal storage disorders (LSDs) and to date there is no cure. Since neurodegeneration is the most relevant pathological feature in MPS-IIIA patients, the treatment of the central nervous system (CNS) lesions represents the goal of any effective therapy for this devastating disorder. During the last years many advances have been made in developing and testing new therapies for brain involvement in MPS-IIIA. These studies have been possible because of the availability of mouse and dog models that recapitulate the MPS-IIIA neuropathological features. Some of these approaches are based on direct CNS administration routes through which the therapeutic molecules access the CNS via the parenchyma (intracerebral injections) or via the cerebrospinal fluid (intraventricular/intrathecal injections). These approaches are highly invasive and poorly suited for clinical use. Minimally invasive approaches are based on systemic injections into the blood stream of therapeutics capable of crossing the blood-brain barrier (BBB). This review will present the background of the clinic and pathology aspects of MPS-IIIA and will describe the current MPS-IIIA preclinical and clinical studies focusing on how a systemic therapeutic strategy based on crossing the BBB has been successfully used to treat CNS pathology and behavioral abnormalities in a mouse model of MPS-IIIA. Future clinical applications of this approach to MPS-IIIA patients will be also discussed together with the possibility of using similar strategies in other LSDs with neurological involvement.

Low-dose, continuous enzyme replacement therapy ameliorates brain pathology in the neurodegenerative lysosomal disorder mucopolysaccharidosis type IIIA.

Repeated replacement of sulphamidase via cerebrospinal fluid injection is an effective treatment for pathological changes in the brain in mice and dogs with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). Investigational trials of this approach are underway in children with this condition, however, infusions require attendance at a specialist medical facility. We sought to comprehensively evaluate the effectiveness of sustained-release (osmotic pump-delivered) enzyme replacement therapy in murine MPS IIIA as this method, if applied to humans, would require only subcutaneous administration of enzyme once the pump was installed. Six-week-old MPS IIIA and unaffected mice were implanted with subcutaneous mini-osmotic pumps connected to an infusion cannula directed at the right lateral ventricle. Either recombinant human sulphamidase or vehicle were infused over the course of 7 weeks, with pumps replaced part-way through the experimental period. We observed near-normalisation of primarily stored substrate (heparan sulphate) in both hemispheres of the MPS IIIA brain and cervical spinal cord, as determined using tandem mass spectrometry. Immunohistochemistry indicated a reduction in secondarily stored GM 3 ganglioside and neuroinflammatory markers. A bias towards the infusion side was seen in some, but not all outcomes. The recombinant enzyme appears stable under pump-like conditions for at least 1 month. Given that infusion pumps are in clinical use in other nervous system disorders, e.g. for treatment of spasticity or brain tumours, this treatment method warrants consideration for testing in large animal models of MPS IIIA and other lysosomal storage disorders that affect the brain. Clinical trials of repeated injection of replacement enzyme into CSF are underway in patients with the inherited neurodegenerative disorder mucopolysaccharidosis type IIIA. In this pre-clinical study, we examined an alternative approach - slow, continual infusion of enzyme using pumps. We observed significant reductions in substrate accumulation and other disease-based lesions in treated mouse brain. Thus, the strategy warrants consideration for testing in large animal models of MPS IIIA and also in other neurodegenerative lysosomal storage disorders.

Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.

Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with pH-sensitive in vitro cytotoxicity in hypoxic MDA-MB-231 and human prostate cancer PC3 cells. Together, we conclude that the HBHAc-HE-based peptide delivery offers a useful means to overcome hypoxia-induced resistance to ADI in breast cancer cells, and to target the mildly acidic tumor microenvironment.

Low-dose, continual enzyme delivery ameliorates some aspects of established brain disease in a mouse model of a childhood-onset neurodegenerative disorder.

AIM: To determine the capacity of continual low-dose lysosomal enzyme infusion into the cerebrospinal fluid of mucopolysaccharidosis type IIIA (MPS IIIA) mice to reverse established neurodegenerative disease. The rationale behind the study is that there is only limited animal model-derived evidence supporting treatment of symptomatic patients, principally because few studies have been designed to examine disease reversibility. METHODS: Twelve-week old MPS IIIA mice were implanted with indwelling unilateral intra-ventricular cannulae. These were connected to subcutaneous mini-osmotic pumps infusing recombinant human sulphamidase. Pump replacement was carried out in some mice at 16-weeks of age, enabling treatment to continue for a further month. Control affected/unaffected mice received vehicle via the same method. Behavioural, neuropathological and biochemical parameters of disease were assessed. RESULTS: Improvement in some, but not all, behavioural parameters occurred. Sulphamidase infusion mediated a statistically significant reduction in primary (heparan sulphate) and secondary (gangliosides GM2, GM3) substrate accumulation in the brain, with small reductions in micro- but not astro-gliosis. There was no change in axonal spheroid number. All mice developed a humoural response, however the antibodies were non-neutralising and no adverse clinical effects were observed. CONCLUSIONS: Continual infusion of replacement enzyme partially ameliorates clinical, histological and biochemical aspects of MPS IIIA mice, when treatment begins at an early symptomatic stage.