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A multicenter, open-label, noncomparative, randomized phase II study (PEPCOL) was conducted to evaluate the efficacy and safety of the irinotecan or PEP02 (MM-398, nanoliposomal irinotecan) with leucovorin (LV)/5-fluorouracil (5-FU) combination as second-line treatment in patients with metastatic colorectal cancer (mCRC). Patients with unresectable mCRC who had failed one prior oxaliplatin-based first-line therapy were randomized toirinotecan with LV/5-FU (FOLFIRI) or PEP02 with LV/5-FU (FUPEP; PEP02 80 mg/m(2) with LV 400 mg/m(2) on day 1 and 5-FU 2400 mg/m(2) on days 1-2). Bevacizumab (5 mg/kg, biweekly) was allowed in both arms. The primary endpoint was 2-month response rate (RR). Fifty-five patients were randomized (FOLFIRI, n = 27; FUPEP, n = 28). In the intent-to-treat population (n = 55), 2-month RR response rate was observed in two (7.4%) and three (10.7%) patients in the FOLFIRI and FUPEP arms, respectively. The most common grade 3-4 adverse events reported in the respective FOLFIRI and FUPEP arms were diarrhea (33% vs. 21%), neutropenia (30% vs. 11%), mucositis (11% vs. 11%), and grade 2 alopecia (26% vs. 25%). FUPEP has activity and acceptable safety profile in oxaliplatin-pretreated mCRC patients.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

This paper describes pressure induced defects on LCD's due to the optical bonding process. It addresses approaches to identify the root cause, as well as process and optical bonding material attributes that have been employed to eliminate defects in commercial applications.

The objective of this study was to define the role of complement activation in the acute and transient toxicities associated with administration of phosphorothioate oligonucleotides in monkeys. In the absence of complement inhibitor, complement activation blocker-2 (CAB-2), i.v. infusion of 20 mg/kg ISIS 2302 produced increases in the concentrations of the complement split products Bb and C5a (100- and 7-fold, respectively). Monkeys also experienced marked changes in bloodpressure (hypertension and hypotension), clinical signs of toxicity (lethargy and periorbital edema), fluctuations in circulating neutrophil counts, and elevations in serum cytokine levels (45-, 12-, and 4-fold increases in IL-6, MCP-1, and IL-12, respectively). Changes occurred at or near the end of infusion and returned to normal over time. One of the three animals died approximately 4 h following infusion of 20 mg/kg ISIS 2302 alone. In contrast, prior treatment with CAB-2 effectively blocked complement activation, as well as the ISIS 2302-induced hemodynamic and clinical responses. Importantly, plasma concentration of ISIS 2302 were unaffected by CAB-2 pretreatment. Thus, the protection afforded by CAB-2 was due to its inhibition of complement activation rather than to any impact on the disposition of ISIS 2302. These results clearly demonstrate the causal relationship between activation of the alternative complement pathway and the hemodynamic and clinical responses associated with rapid infusion of phosphorothioate oligonucleotides. Demonstration of this relationship underscores the importance of avoiding complement activation in patients to ensure the continued safe use of phosphorothioate oligodeoxynucleotides.

Complement plays a critical role in many pathologic processes and in xenograft rejection. Therefore, effective complement inhibitors are of great interest. In pig-to-primate organ transplantation, hyperacute rejection results from antibody deposition and complement activation. Complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF) and membrane cofactor protein, inhibits C3 and C5 convertases of both classical and alternative pathways. CAB-2 reduces complement-mediated tissue injury of a pig heart perfused ex vivo with human blood. Therefore, we studied the efficacy of CAB-2 when a pig heart is transplanted heterotopically into rhesus monkeys receiving no immunosuppression. Graft survival in three control monkeys was 1.26 +/- 0.2 h; it was markedly prolonged in eight monkeys that received CAB-2. Of the six monkeys that received a single dose of CAB-2 (15 mg/kg i.v.), four had graft survivals of 21, 95, 96, and 108 h, and two died at 7 to 11 h post-transplant with a beating graft, as a result of technical complications. The two monkeys given multiple doses of CAB-2 had graft survivals of 95 and 96 h. CAB-2 markedly inhibited complement activation, as shown by a strong reduction in generation of C3a and SC5b-9. At graft rejection, tissue deposition of iC3b, C4 and C9 was similar or slightly reduced from controls, and deposition of IgG, IgM, C1q and fibrin did not change. Thus, complement inhibition with CAB-2 abrogates hyperacute rejection of pig hearts transplanted into rhesus monkeys, but does not prevent delayed/acute vascular rejection. These studies demonstrate that the beneficial effects of complement inhibition on survival of a pig heart xenograft in rhesus monkeys are similar to those in other primate species and that CAB-2 may be useful in xenotransplantation and other complement-mediated conditions.

CMI-977 is a potent 5-lipoxygenase inhibitor that intervenes in the production of leukotrienes and is presently being developed for the treatment of chronic asthma. It is a single enantiomer with an all-trans (2S,5S) configuration. Of the four isomers of CMI-977, the S,S isomer was found to have the best biological activity and was selected for further development. The enantiomerically pure product was synthesized on a 2-kg scale from (S)-(+)-hydroxymethyl-γ-butyrolactone.

Complement plays a critical role in many pathologic processes and in xenograft rejection. Therefore, effective complement inhibitors are of great interest. In pig-to-primate organ transplantation, hyperacute rejection results from antibody deposition and complement activation. Complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF) and membrane cofactor protein, inhibits C3 and C5 convertases of both classical and alternative pathways. CAB-2 reduces complement-mediated tissue injury of a pig heart perfused ex vivo with human blood. Therefore, we studied the efficacy of CAB-2 when a pig heart is transplanted heterotopically into rhesus monkeys receiving no immunosuppression. Graft survival in three control monkeys was 1.26 +/- 0.2 h; it was markedly prolonged in eight monkeys that received CAB-2. Of the six monkeys that received a single dose of CAB-2 (15 mg/kg i.v.), four had graft survivals of 21, 95, 96, and 108 h, and two died at 7 to 11 h post-transplant with a beating graft, as a result of technical complications. The two monkeys given multiple doses of CAB-2 had graft survivals of 95 and 96 h. CAB-2 markedly inhibited complement activation, as shown by a strong reduction in generation of C3a and SC5b-9. At graft rejection, tissue deposition of iC3b, C4 and C9 was similar or slightly reduced from controls, and deposition of IgG, IgM, C1q and fibrin did not change. Thus, complement inhibition with CAB-2 abrogates hyperacute rejection of pig hearts transplanted into rhesus monkeys, but does not prevent delayed/acute vascular rejection. These studies demonstrate that the beneficial effects of complement inhibition on survival of a pig heart xenograft in rhesus monkeys are similar to those in other primate species and that CAB-2 may be useful in xenotransplantation and other complement-mediated conditions.

Background —We previously demonstrated that inhibiting formation of terminal complement components (C5a and C5b-9) prevents platelet and neutrophil (PMN) but not monocyte activation during simulated extracorporeal circulation (SECC). This study examined whether earlier complement inhibition during SECC, blocking C3a formation, would additionally prevent monocyte activation. Methods and Results —SECC was established by recirculating heparinized whole blood from human volunteers on a membrane oxygenator. CAB-2, a chimeric protein constructed from genes encoding the complement regulatory proteins CD46 and CD55, inactivates the C3/C5 convertases and blocks in vitro generation of C3a, C5a, and C5b-9. CAB-2 was used in 4 experiments at a final concentration of 300 μg/mL and 4 experiments at 30 μg/mL; 4 control runs used vehicle alone. Samples were assayed for C3a and C5b-9, monocyte activation (CD11b upregulation), PMN activation (CD11b upregulation and elastase release), and platelet activation (P-selectin expression and monocyte-platelet conjugate formation). CAB-2 at both doses significantly inhibited formation of C3a and C5b-9 during SECC. High-dose CAB-2 significantly blocked monocyte and PMN CD11b upregulation and PMN elastase release. CAB-2 also inhibited formation of platelet activation–dependent monocyte–platelet conjugates. Conclusions —Blockade of complement activation early in the common pathway inhibited monocyte CD11b upregulation during SECC, suggesting that early complement components contribute most to monocyte activation during SECC. As expected, PMN and platelet activation were blocked by terminal complement inhibition. This investigation further elucidates the relation between complement and blood cell activation during simulated cardiopulmonary bypass.

613 Background: PEP02 (MM-398) is a nanoliposomal formulation of irinotecan (CPT-11) that has improved pharmacokinetics (PK) and tumor distribution of CPT-11 and its active metabolite, SN-38. PEP02 single agent q3w has shown preliminary efficacy and safety in Phase II pancreatic and gastric cancer studies. Since irinotecan is approved for metastatic colorectal cancer (mCRC) and biweekly regimens are widely used, the aims of this study are to determine the maximum tolerated dose (MTD), characterize the PK and pharmacogenetics (PGx), and explore the efficacy of PEP02 q2w in mCRC. Methods: Patients (pts) with disease progression after 1st-line oxaliplatin-based chemotherapy, ECOG PS 0-1, and without prior exposure to irinotecan were eligible. PEP02 was given on day 1 and 15 of each 28 day treatment cycle. The starting dose was 80 mg/m2 and escalated by 10 mg/m2 to the target dose of 100 mg/m2. PK was evaluated during the 1st cycle and the tumor response was assessed by RECIST. Results: A total of 18 pts (M/F 9/9; median age 57.5) were enrolled, with 6 at each dose level. Dose-limiting toxicity manifested as G3 diarrhea was observed in one pt per dose level. The target dose of 100 mg/m2 was determined to be the MTD. Nine pts had dose delayed (4, 3, 2 at 80, 90, 100 mg/m2), mostly because of neutropenia. The PK and PGx are being analysed. As of August 2011, there are 3 pts still on study treatment and 17 pts evaluable for tumor response. Four pts (2 at 80 mg/m2, 1 each at 90 and 100 mg/m2) showed partial response (3 after 2 cycles and 1 after 8 cycles) and 8 pts (3 each at 80 and 90 mg/m2, 2 at 100 mg/m2) maintained stable disease for at least 2 cycles, which resulted in a response rate (RR) of 23.5% and a disease control rate (DCR) of 70.6%. Current median progression-free survival (PFS) is 4 months and 8 pts (47%) had PFS ≥ 6 months. Conclusions: The MTD of biweekly PEP02 is 100 mg/m2. As a 2nd-line monotherapy after oxaliplatin-based chemotherapy, the efficacy results indicate the potential benefit of PEP02 for mCRC (FOLFIRI-1 achieved only 4% RR, 34% DCR, and 2.5 months PFS in FOLFOX pretreated pts). A randomized Phase II study evaluating PEP02 plus 5-FU/LV (FUPEP regimen) vs. FOLFIRI is currently ongoing in France.

MM-398 (aka PEP02) is a stable, nanotherapeutic encapsulation of the pro-drug CPT-11 (irinotecan) that is currently in clinical development. In preclinical experiments, treatment with MM-398 resulted in significantly higher intratumoral concentrations of both irinotecan (142-fold) and its major metabolite, SN-38 (9-fold) and exhibited superior anti-tumor activity compared to free irinotecan in multiple tumor xenografts. Subsequently, multiple phase 1 and 2 studies have established a pharmacokinetic and safety profile that supports continued clinical development, including in pancreatic, gastric, colorectal and potentially other cancers. Because current evidence suggests that resistance to pancreatic cancer is driven largely by inadequate drug penetration into these often poorly vascularized, stromally dense and hypoxic tumors, we sought to better understand how this relatively large (100nm) liposomal nanotherapeutic could potentially result in increased efficacy in very advanced gemcitabine-resistant pancreatic cancer and other cancer types. We developed a mechanism-based PK model of MM-398 and free irinotecan designed to predict intratumor SN-38 levels. Sensitivity analysis revealed that for MM-398 local activation of irinotecan to SN-38 was a far more important parameter than systemic activation. Through cellular uptake studies, we demonstrated in vitro that MM-398 was preferentially internalized by phagocytic macrophage/monocyte cell lines and, to a far lesser extent, by tumor cell lines. Furthermore, tumor microdistribution studies by flow cytometry and IHC showed uptake of MM-398 liposomes in both tumor cells and tumor-associated macrophages with more liposomal material being present in the macrophages. This distribution also suggests that macrophages may contribute to the postulated rate limiting process of irinotecan activation. The sensitivity analysis also suggested that tumor permeability and vascularization are important determinants of tumor-associated SN-38 levels for both free irinotecan and MM-398. To determine the effect of MM-398 on these parameters we treated mice bearing HT29 (colorectal cancer) xenografts with a single dose of MM-398 and measured hypoxic markers (CAIX) and microvessel density (CD31) by IHC. Tumors treated with MM-398 showed a greater degree of CD31 staining and lower CAIX staining, indicating that MM-398 may be able to affect tumor characteristics that traditionally have contributed to therapy resistance and limited the delivery of cancer therapeutics and resistance. In summary, encapsulation of irinotecan alters rate-limiting processes that determine tumoral SN-38 levels. Delivery of MM-398 is believed to alter tumor microvessel density and decrease hypoxia. These intriguing mechanisms of action findings support the continued clinical development of MM-398 as a differentiated therapeutic for several cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C207.

6 Background: PEP02 is a novel nanoparticle liposome formulation of irinotecan (CPT-11). In phase I studies, PEP02 has improved pharmacokinetics (PK) of CPT-11 and its active metabolite-SN38 with encouraging safety and tumor response in several cancer types including gastric cancer. This study evaluated the efficacy and safety of PEP02 (P), irinotecan (I) or docetaxel (D) as a single agent in gastric or gastroesophageal junction (GEJ) adenocarcinoma. Methods: A randomized, 3 arms (1:1:1), Simon's 2-stage (2/21, 5/41 based on tumor response) study was conducted in Europe and Asia. Patients (pts) with locally advanced or metastatic disease, failed to one prior chemotherapy, ECOG PS ≤ 2, at least 1 measurable lesion, no prior CPT-11 or taxane, were treated with P - 120 mg/m2, I - 300 mg/m2, or D - 75 mg/m2 every 3 weeks. PK and pharmacogenetics (PGx) samples were collected for pts in P and I arms. Results: A total of 135 pts were randomized with 132 (44 per arm) treated between Jan 2008 and Jun 2010. Pts demographics (P/I/D): median age: 56/62/58, male (%): 79.5/77.3/77.3, Pts from Europe (%): 54.6/52.3/56.8, metastatic (%): 97.7/90.9/97.7, gastric adenocarcinoma (%): 84.1/79.6/68.2, and ECOG 0 + 1 (%): 93.2/93.2/90.9. The confirmed responders of P/I/D were 6 (13.6%)/3 (6.8%)/7 (15.9%) and disease control were 27 (61.4%)/27 (61.4%)/24 (54.6%). These three arms have similar progression free survival and overall survival. If stratified by region, Asian pts had longer survival than European pts. Toxicities of P/I/D were: grade 3/4 neutropenia (%): 9.1/13.6/15.9. grade 3/4 diarrhea (%): 27.3/18.2/2.3, hand-foot syndromes (%): 0.0/6.8/18.2. It was notable that symptoms related to acute cholinergic syndrome were less reported in P arm than in I arm. The PK data showed the mean T1/2, Cmax and AUC0→∞ of SN-38 in P/I arms were 88.8/22.8 hr, 8.79/44.1 ng/mL and 879/440 hr x ng/mL. Conclusions: This randomized phase II study suggests that PEP02 improves the PK profile and tumor response over irinotecan, and it is as efficacious as docetaxel in the 2nd-line treatment for gastric or GEJ adenocarcinoma. PEP02 is worthy of further evaluation as either 1st- or 2nd-line setting in future gastric cancer studies. [Table: see text]

In the guinea pig-to-rat model of hyperacute xenograft (Xg) rejection, the effect of complement inhibition using systemically administered soluble complement receptor type 1 (sCR1) on discordant cardiac Xg survival was investigated. In PBS-treated control Xg recipients (n = 13), hyperacute rejection was rapid, with a mean Xg survival of 17 +/- 4 min. Therapy with sCR1 prolonged survival of cardiac Xgs in a dose-dependent manner. A 3 mg/kg bolus of sCR1 (n = 4) prolonged Xg survival to 64 +/- 29 min (not significant). Increasing the sCR1 dose to 5.9 mg/kg (n = 4) significantly delayed Xg rejection to 71 +/- 17 min (P-0.026, log-rank test vs. control). In 10 recipients treated with 15 mg/kg sCR1, mean Xg survival was further prolonged to 189 +/- 36 min (P-0.0004) with no adverse effects. While 2 of 8 recipients receiving 60 mg/kg sCR1 died with functioning Xgs at 30 and 300 min due to anastomotic bleeding, Xg survival averaged over 12 hr (747 +/- 100 min, P-0.0004) in the remaining 6 recipients. sCR1 administration significantly inhibited serum complement activity in a parallel dose-dependent fashion, with the 60 mg/kg dose reducing complement activity by 95 +/- 1 and 96 +/- 1% five and 30 min following Xg reperfusion, respectively. Immunofluorescence microscopy revealed rat IgM bound to all cardiac Xgs in control as well as sCR1-treated recipients. In addition, serial histologic examination of cardiac Xgs harvested within 21 min of graft reperfusion revealed occlusive platelet aggregates within the coronary vessels as well as interstitial hemorrhage and myocardial necrosis in Xgs from control recipients, all of which were only minimally present in Xgs from recipients treated with sCR1. These studies show that complement inhibition with sCR1 significantly delays hyperacute cardiac Xg rejection in this discordant model and may be an important component in a therapeutic protocol for xenotransplantation.

Background PEP02 (also known as MM-398, nal-IRI) is a novel nanoparticle formulation of irinotecan encapsulated in liposomes. The aims of this study were to investigate the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and pharmacokinetics (PK) of PEP02 in combination with 5-FU and LV, in patients with advanced refractory solid tumors. Methods Patients were enrolled in cohorts to receive PEP02 from 60 to 120 mg/m2 (dose expressed as the irinotecan hydrochloride trihydrate salt) as a 90-min intravenous infusion on day 1, followed by 24 h infusion of 5-FU 2,000 mg/m2 and LV 200 mg/m2 on days 1 and 8, every 3 weeks. Results A total of 16 patients were assigned to four dose levels, 60 (three patients), 80 (six patients), 100 (five patients) and 120 mg/m2 (two patients). DLT was observed in four patients, two at the 100 mg/m2 dose level (one had grade III infection with hypotension and grade III hemorrhage; the other had grade III diarrhea and grade IV neutropenia), and two at the 120 mg/m2 dose level (one had grade III diarrhea and grade IV neutropenia; the other had grade III diarrhea). The MTD of PEP02 was determined as 80 mg/m2. The most common treatment-related adverse events were nausea (81%), diarrhea (75%) and vomiting (69%). Among the six patients who received the MTD, one patient exhibited partial response, four patients had stable disease and one showed progressive disease. Pharmacokinetic data showed that PEP02 had a lower peak plasma concentration, longer half-life, and increased area under the plasma concentration-time curve from zero to time t of SN-38 than irinotecan at similar dose level. Conclusions The MTD of PEP02 on day 1 in combination with 24-h infusion of 5-FU and LV on days 1 and 8, every 3 weeks was 80 mg/m2, which will be the recommended dose for future studies. Trial registration The trial was retrospectively registered (NCT02884128) with date of registration: August 12, 2016. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2933-6) contains supplementary material, which is available to authorized users.