Mussel adhesive proteins (MAPs) have drawn interest for their ability to form strong adhesive bonds to a variety of substrates in wet environments. 1 There have been efforts to develop biomedical adhesives from these proteins, yet this has been hampered by the difficulty of isolation from biological sources. 2 Synthetic polymers are a potential source of functionally equivalent adhesives, yet little is known about how the MAPs function and therefore what must be incorporated into a synthetic analogue. The catechol functionality of L-3,4-dihydroxyphenylalanine, DOPA, residues is thought to be responsible for adhesion and cross-linking of the proteins; however, the mechanisms for these processes are unknown. The potential involvement of L-lysine and other polar residues in these reactions further complicates analytical efforts. 1 Speculation on the key components of these materials has been limited since no cross-link bond or specific bond to a substrate has been identified as yet. Through analysis of amino acid derivatives and simple copolypeptides under adhesive curing conditions, we have determined that DOPA is the only functional element required to reproduce the properties of MAPs. Furthermore, the primary roles of both catechol and o-quinone forms of DOPA can be assigned to adhesive bonding and cross-link formation, respectively.The most detailed studies on MAPs have focused on the blue mussel, Mytilus edulis. This organism anchors itself to surfaces by means of plaques on the ends of fibrous threads. A considerable variety of adhesive proteins have been isolated from uncured plaques. These proteins range in mass from ca. 5 to 120 kDa, and all contain high levels of DOPA (ca. 5-20 mol %). 3 Variants also contain elevated levels of other polar amino acids such as hydroxylated prolines, lysine, and 4-hydroxyarginine. The variability of these proteins, in terms of their chain lengths, sequences, and compositions, has made it difficult to identify the important components responsible for adhesion.It is known that catechol oxidase enzymes are present in MAP secretions that convert the catechol groups of DOPA into highly reactive o-quinone functionalities. 4 Numerous reactions have been proposed for cross-linking of the quinones (Scheme 1), yet none of these have been experimentally verified in MAPs. 1 The most often cited reaction is the Michael addition of side-chain amino groups of lysine residues to a DOPA-quinone residue. 5 Although all attempts to detect this product to date have been unsuccessful, the importance of the Michael addition in quinone chemistry has generated strong support for lysine cross-linking in MAPs. 6 Our goal was to experimentally identify the roles of amino acids that are active in the adhesive chemistry of MAPs. Problems

IT-101, a cyclodextrin polymer-based nanoparticle containing camptothecin, is in clinical development for the treatment of cancer. Multiorgan pharmacokinetics and accumulation in tumor tissue of IT-101 is investigated by using PET. IT-101 is modified through the attachment of a 1,4,7,10-tetraazacyclododecane-1,4,7-Tris-acetic acid ligand to bind 64 Cu 2؉ . This modification does not affect the particle size and minimally affects the surface charge of the resulting nanoparticles. PET data from 64 Cu-labeled IT-101 are used to quantify the in vivo biodistribution in mice bearing Neuro2A s.c. tumors. The 64 Cu-labeled IT-101 displays a biphasic plasma elimination. Approximately 8% of the injected dose is rapidly cleared as a low-molecular-weight fraction through the kidneys. The remaining material circulates in plasma with a terminal half-life of 13.3 h. Steadily increasing concentrations, up to 11% injected dose per cm 3 , are observed in the tumor over 24 h, higher than any other tissue at that time. A 3-compartment model is used to determine vascular permeability and nanoparticle retention in tumors, and is able to accurately represent the experimental data. The calculated tumor vascular permeability indicates that the majority of nanoparticles stay intact in circulation and do not disassemble into individual polymer strands. A key assumption to modeling the tumor dynamics is that there is a ''sink'' for the nanoparticles within the tumor. Histological measurements using confocal microscopy show that IT-101 localizes within tumor cells and provides the sink in the tumor for the nanoparticles.cancer ͉ camptothecin ͉ cyclodextrin ͉ polymer ͉ intracellular delivery C hemotherapeutics are the mainstay of cancer treatment for advanced and/or metastatic tumors. However, their effectiveness is typically limited by toxicity in healthy, normal tissues with rapidly dividing cells, such as bone marrow or the gastrointestinal tract. One approach to increase the therapeutic index of chemotherapeutics is site-directed delivery by using various carrier systems. Numerous types of delivery technologies have been developed for this purpose, such as liposomes (1, 2), conjugates with antibodies or small molecules targeted to tumor antigens (3, 4), and macromolecular polymer carriers (5). Carrier-drug composites are nanoscaled therapeutics, and nanoparticle cancer therapeutics that have been used in humans have been reviewed elsewhere (6).The use of nanoscaled therapeutics takes advantage of the unique tumor physiology characterized by a high density of abnormal blood vessels, high vascular permeability, and decreased rate of clearance due to a lack of lymphatic drainage, all of which act together to cause accumulation of macromolecules through the enhanced permeability and retention (EPR) effect (7). The magnitude of this effect is affected by a number of parameters that are either host-related (tumor perfusion, vascularity, vascular permeability) or nanoparticle-related (plasma half-life, clearance, hydrodynamic size, surface char...

Preclinical efficacy of i.v. IT-101, a nanoparticulate conjugate of 20(S)-camptothecin and a cyclodextrin-based polymer, was investigated in several mouse xenografts.The effects of different multiple dosing schedules on tumor growth of LS174Tcolon carcinoma xenografts are elucidated. All multiple dosing schedules administered over15 to19 days resulted in enhanced efficacy compared with untreated or single-dose groups. Further improvements in antitumor efficacy were not observed when the dosing frequency was increased from three weekly doses to five doses at 4-day intervals or 5 days of daily dosing followed by 2 days without dosing repeated in three cycles using similar cumulative doses. This observation was attributed to the extended release characteristics of camptothecin from the polymer. Antitumor efficacy was further evaluated in mice bearing six different s.c. xenografts (LS174T and HT29 colorectal cancer, H1299 nonŝ mall-cell lung cancer, H69 small-cell lung cancer, Panc-1pancreatic cancer, and MDA-MB-231 breast cancer) and one disseminated xenograft (TC71-luc Ewing's sarcoma). In all cases, a single treatment cycle of three weekly doses of IT-101resulted in a significant antitumor effect. Complete tumor regression was observed in all animals bearing H1299 tumors and in the majority of animals with disseminated Ewing's sarcoma tumors. Importantly, IT-101is effective in a number of tumors that are resistant to treatment with irinotecan (MDA-MB-231, Panc-1, and HT29), consistent with the hypothesis that polymeric drug conjugates may be able to overcome certain kinds of multidrug resistance. Taken together, these results indicate that IT-101 has good tolerability and antitumor activity against a wide range of tumors.

Summary Patients with advanced solid malignancies were enrolled to an open-label, single-arm, dose-escalation study, in which CRLX101 was administered intravenously over 60 min among two dosing schedules, initially weekly at 6, 12, and 18 mg/m2 and later bi-weekly at 12, 15, and 18 mg/m2. The maximum tolerated dose (MTD) was determined at 15 mg/m2 bi-weekly, and an expansion phase 2a study was completed. Patient samples were obtained for pharmacokinetic (PK) and pharmacodynamic (PD) assessments. Response was evaluated per RECIST criteria v1.0 every 8 weeks. Sixty-two patients (31 male; median age 63 years, range 39-79) received treatment. Bi-weekly dosing was generally well tolerated with myelosuppression being the dose-limiting toxicity. Among all phase 1/2a patients receiving the MTD (n=44), most common grade 3/4 adverse events were neutropenia and fatigue. Evidence of systemic plasma exposure to both the polymer-conjugated and unconjugated CPT was observed in all treated patients. Mean elimination unconjugated CPT Tmax values ranged from 17.7 to 24.5 h, and maximum plasma concentrations and areas under the curve were generally proportional to dose for both polymer-conjugated and unconjugated CPT. Best overall response was stable disease in 28 patients (64 %) treated at the MTD and 16 (73 %) of a subset of NSCLC patients. Median progression-free survival (PFS) for patients treated at the MTD was 3.7 months and for the subset of NSCLC patients was 4.4 months. These combined phase 1/2a data demonstrate encouraging safety, pharmacokinetic, and efficacy results. Multinational phase 2 clinical development of CRLX101 across multiple tumor types is ongoing.

Nanoparticles are currently being investigated in a number of human clinical trials. As information on how nanoparticles function in humans is difficult to obtain, animal studies that can be correlative to human behavior are needed to provide guidance for human clinical trials. Here, we report correlative studies on animals and humans for CRLX101, a 20-to 30-nm-diameter, multifunctional, polymeric nanoparticle containing camptothecin (CPT). CRLX101 is currently in phase 2 clinical trials, and human data from several of the clinical investigations are compared with results from multispecies animal studies. The pharmacokinetics of polymer-conjugated CPT (indicative of the CRLX101 nanoparticles) in mice, rats, dogs, and humans reveal that the area under the curve scales linearly with milligrams of CPT per square meter for all species. Plasma concentrations of unconjugated CPT released from CRLX101 in animals and humans are consistent with each other after accounting for differences in serum albumin binding of CPT. Urinary excretion of polymer-conjugated CPT occurs primarily within the initial 24 h after dosing in animals and humans. The urinary excretion dynamics of polymer-conjugated and unconjugated CPT appear similar between animals and humans. CRLX101 accumulates into solid tumors and releases CPT over a period of several days to give inhibition of its target in animal xenograft models of cancer and in the tumors of humans. Taken in total, the evidence provided from animal models on the CRLX101 mechanism of action suggests that the behavior of CRLX101 in animals is translatable to humans.nanomedicine | clinical translation | interspecies scaling | pharmacodynamics | Nanoparticles

BACKGROUND: Only a few clinical trials have been conducted in patients with advanced pancreatic cancer after failure of first-line gemcitabine-based chemotherapy. Therefore, there is no current consensus on the treatment of these patients. We conducted a randomised phase II study of the modified FOLFIRI.3 (mFOLFIRI.3; a regimen combining 5-fluorouracil (5-FU), folinic acid, and irinotecan) and modified FOLFOX (mFOLFOX; a regimen combining folinic acid, 5-FU, and oxaliplatin) regimens as second-line treatments in patients with gemcitabine-refractory pancreatic cancer. METHODS: The primary end point was the 6-month overall survival rate. The mFOlFIRI.3 regimen consisted of irinotecan (70 mg m À2 ; days 1 and 3), leucovorin (400 mg m À2 ; day 1), and 5-FU (2000 mg m À2 ; days 1 and 2) every 2 weeks. The mFOLFOX regimen was composed of oxaliplatin (85 mg m À2 ; day 1), leucovorin (400 mg m À2 ; day 1), and 5-FU (2000 mg m À2 ; days 1 and 2) every 2 weeks. RESULTS: Sixty-one patients were randomised to mFOLFIRI.3 (n ¼ 31) or mFOLFOX (n ¼ 30) regimen. The six-month survival rates were 27% (95% confidence interval (CI) ¼ 13 -46%) and 30% (95% CI ¼ 15 -49%), respectively. The median overall survival periods were 16.6 and 14.9 weeks, respectively. Disease control was achieved in 23% (95% CI ¼ 10 -42%) and 17% patients (95% CI ¼ 6 -35%), respectively. The number of patients with at least one grade 3/4 toxicity was identical (11 patients, 38%) in both groups: neutropenia (7 patients under mFOLFIRI.3 regimen vs 6 patients under mFOLFOX regimen), asthaenia (1 vs 4), vomiting (3 in both), diarrhoea (2 vs 0), and mucositis (1 vs 2). CONCLUSION: Both mFOLFIRI.3 and mFOLFOX regimens were tolerated with manageable toxicity, offering modest activities as second-line treatments for patients with advanced pancreatic cancer, previously treated with gemcitabine.

We have synthesized methylated mono- and di(ethylene glycol)-functionalized polymers of L-serine and L-cysteine that adopt beta-sheet conformations in the solid state: poly(O-(2-(2-methoxyethoxy)ethyl)-L-serine), poly(1); poly(O-(2-(methoxy)ethyl)-L-serine), poly(2); and poly(S-(2-(2-methoxyethoxy)ethoxy)carbonyl-L-cysteine), poly(3). Of these three polymers, only poly(1) was found to be highly soluble in water independent of pH. Circular dichroism analysis of poly(1) in water or trifluoroethanol at 25 degrees C revealed that it is in a random conformation, which was unperturbed by changes in pH, buffer, or temperature. However, addition of methanol or acetonitrile to aqueous solutions of poly(1) resulted in a transition to the beta-sheet conformation, as found in the solid state. The polymers were synthesized by transition metal catalyzed polymerization of amino acid-N-carboxyanhydrides, prepared from the functionalized amino acids and represent a new class of readily processable beta-sheet forming polypeptides.

No abstract