Cyclodextrin-based Polymer Research Articles

Pharmacodynamic and pharmacogenomic study of the nanoparticle conjugate of camptothecin CRLX101 for the treatment of cancer

CRLX101 is a nanopharmaceutical consisting of cyclodextrin-based polymer molecule and camptothecin. The CRLX101 nanoparticle is designed to concentrate and slowly release camptothecin in tumors over an extended period of time. Tumor biopsy and blood samples collected from patients with advanced solid malignancies before and after CRLX101 treatment are subjected to immunohistochemistry and pharmacogenomics. The expression of Topoisomerase-1, Ki-67, CaIX, CD31 and VEGF decreased after CRLX101 treatment. The expressions of these proteins are inversely proportional with survival duration of the patients. The Drug Metabolism Enzymes and Transporters (DMET) array shows an allele frequency in patients similar to global populations with none of the SNPs associated with toxicity. The results suggest that the observed lower toxicity is not likely to be due to different genotypes in SNPs. CRLX101 demonstrates a promising anti-tumor activity in heavily pre-treated or treatment-refractory solid tumor malignancies presumably by inhibition of proliferation and angiogenesis correlating with tumor growth inhibition. From the Clinical EditorIn this cancer treatment study clinical samples collected from patients were subjected to immunohistochemistry and pharmacogenomics. The expressions of key proteins that are inversely proportional with survival duration of the patients decreased after treatment with CRLX101, a camptothecin slow-release nanoparticle conjugate. This anti-tumor activity in heavily pre-treated and treatment resistant solid tumors, promises a novel therapeutic approach.

Porous silicon-cyclodextrin based polymer composites for drug delivery applications

One of the main applications of porous silicon (PSi) in biomedicine is drug release, either as a single material or as a part of a composite. PSi composites are attractive candidates for drug delivery systems because they can display new chemical and physical characteristics, which are not exhibited by the individual constituents alone. Since cyclodextrin-based polymers have been proven efficient materials for drug delivery, in this work β-cyclodextrin–citric acid in-situ polymerization was used to functionalize two kinds of PSi (nanoporous and macroporous). The synthesized composites were characterized by microscopy techniques (SEM and AFM), physicochemical methods (ATR-FTIR, XPS, water contact angle, TGA and TBO titration) and a preliminary biological assay was performed. Both systems were tested as drug delivery platforms with two different model drugs, namely, ciprofloxacin (an antibiotic) and prednisolone (an anti-inflammatory), in two different media: pure water and PBS solution. Results show that both kinds of PSi/β-cyclodextrin–citric acid polymer composites, nano- and macro-, provide enhanced release control for drug delivery applications than non-functionalized PSi samples.

Vibrational Density of States and Elastic Properties of Cross-Linked Polymers: Combining Inelastic Light and Neutron Scattering

The vibrational dynamics of a new class of cross-linked polymers made up of cyclodextrins is here investigated in the microscopic range by the joint use of light and inelastic neutron scattering experiments. The effect of increasing the connectivity of the polymeric network on the vibrational dynamics of the system is studied by exploiting the complementarity of these two different probes. The derived densities of vibrational states of the polymers evidence the presence of the characteristic anomalous excess of vibrational modes with respect to the Debye level, already observed in the low-frequency Raman spectra and referred to as boson peak (BP). The overall analysis of the spectra suggests an emerging picture in which the motions of hydrogen atoms of the polymers are progressively hampered when the cross-linking degree of the covalent network increases. At the same time, the frequency and intensity of the BP are found to significantly change by increasing the cross-linking degree of the polymeric network, as clearly suggested by the existence of a scaling-law for the BP evolution. These findings support the conclusion that the growing of the covalent connectivity of the system induces a general modifications of the elastic properties of these cyclodextrin-based polymers, which are, once again, modulated by the cross-linking agent/cyclodextrin molar ratio.

Serial Diffusion MRI to Monitor and Model Treatment Response of the Targeted Nanotherapy CRLX101

Targeted nanotherapies are being developed to improve tumor drug delivery and enhance therapeutic response. Techniques that can predict response will facilitate clinical translation and may help define optimal treatment strategies. We evaluated the efficacy of diffusion-weighted magnetic resonance imaging to monitor early response to CRLX101 (a cyclodextrin-based polymer particle containing the DNA topoisomerase I inhibitor camptothecin) nanotherapy (formerly IT-101), and explored its potential as a therapeutic response predictor using a mechanistic model of tumor cell proliferation. Diffusion MRI was serially conducted following CRLX101 administration in a mouse lymphoma model. Apparent diffusion coefficients (ADCs) extracted from the data were used as treatment response biomarkers. Animals treated with irinotecan (CPT-11) and saline were imaged for comparison. ADC data were also input into a mathematical model of tumor growth. Histological analysis using cleaved-caspase 3, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, Ki-67, and hematoxylin and eosin (H&E) were conducted on tumor samples for correlation with imaging results. CRLX101-treated tumors at day 2, 4, and 7 posttreatment exhibited changes in mean ADC = 16 ± 9%, 24 ± 10%, 49 ± 17%, and size (TV) = -5 ± 3%, -30 ± 4%, and -45 ± 13%, respectively. Both parameters were statistically greater than controls [p(ADC) ≤ 0.02, and p(TV) ≤ 0.01 at day 4 and 7], and noticeably greater than CPT-11-treated tumors (ADC = 5 ± 5%, 14 ± 7%, and 18 ± 6%; TV = -15 ± 5%, -22 ± 13%, and -26 ± 8%). Model-derived parameters for cell proliferation obtained using ADC data distinguished CRLX101-treated tumors from controls (P = 0.02). Temporal changes in ADC specified early CRLX101 treatment response and could be used to model image-derived cell proliferation rates following treatment. Comparisons of targeted and nontargeted treatments highlight the utility of noninvasive imaging and modeling to evaluate, monitor, and predict responses to targeted nanotherapeutics.

シクロデキストリンポリマーのリンゴジュース中のパツリン分析への利用

パツリン産生菌の一種である Penicillium expansum はリンゴ腐敗菌でもあり，リンゴおよびリンゴ製品におけるパツリン汚染の原因菌として知られている．リンゴジュース中のパツリンの分析法としては，酢酸エチルを用いた液液抽出後， HPLC-UV もしくは LC-MS を用いて分析する方法が広く用いられている．これまでの研究において，シクロデキストリンポリマーが様々な有機物の混在するリンゴジュース中においてパツリンを吸着することが示されている．本研究では，4,4'-methylenebis （phenyl isocyanate）をシクロデキストリンの架橋剤として用い合成したポリマーをリンゴジュース中のパツリンの固相抽出に利用することを検討した．固相カラムのコンディショニングに用いる溶媒，固相カラムへの分析試料の負荷容量，洗浄液の溶媒，溶離液の溶媒，容量を検討し，パツリン分析の最適化を行った．最適化した方法において，10，20，50，80，100 ng mL－1 のパツリン添加試料におけるパツリンの回収率（相対標準偏差）はそれぞれ 78（20）%，71（13）%，78（17）%，71（7.1）%，67（2.9）%であった．リンゴジュース中のパツリンの本分析法における定量下限（LOQ）は 10ng mL－1 であった．

Photoinduced Fluorescence Activation and Nitric Oxide Release with Biocompatible Polymer Nanoparticles

A viable strategy to encapsulate a fluorophore/photochrome dyad and a nitric oxide photodonor within supramolecular assemblies of a cyclodextrin-based polymer in water was developed. The two photoresponsive guests do not interact with each other within their supramolecular container and can be operated in parallel under optical control. Specifically, the dyad permits the reversible switching of fluorescence on a microsecond timescale for hundreds of cycles, and the photodonor enables the irreversible release of nitric oxide. Furthermore, these supramolecular assemblies cross the membrane of human melanoma cancer cells and transport their cargo in the cytosol. The fluorescence of one component allows the visualization of the labeled cells, and its switchable character could, in principle, be used to acquire super-resolution images, while the release of nitric oxide from the other induces significant cell mortality. Thus, our design logic for the construction of biocompatible nanoparticles with dual functionality might evolve into the realization of valuable photoresponsive probes for imaging and therapeutic applications.

Translating Nanotechnology from Bench to Pharmaceutical Market: Barriers, Success, and Promises

Nanotechnology is a buzzword of this millennium and it has transformed the face of research in science and technology. The advent of nanotechnology has also influenced the biomedical and pharmaceutical research since last decade. Various nano-architectures have been designed for improving the therapeutic performance of drugs, proteins, peptides, and genes and to achieve their targeting at the site of action. Although nanotechnology has demonstrated dramatic potential in drug delivery research, like any technology, its real success depends on the ability of drug delivery scientists to translate and scale innovations to the commercial pharmaceutical products. It is indeed a very challenging task to successfully overcome manufacturing, clinical, and regulatory hurdles associated with a nanotech product. Nevertheless, the pharmaceutical industry has witnessed commercialization of the nanotechnology-based products for various applications. In the present special issue, we have tried to consolidate various aspects of existing and upcoming nanotechnologies for drug delivery. Contribution by V. Morigi et al. takes an overview of business potential and market trend of pharmaceutical nanotechnology. The authors have also discussed financial aspects of nanotechnology by citing noteworthy examples of few nanotech products that have already been commercialized. This contribution could be useful to scientists aiming to start up nanotechnological business ventures. Contribution by N. Anton et al. demonstrates how nanotechnology can change the face of conventional drug delivery systems. In this interesting investigation, the authors demonstrate that coating of conventional tablets with lipid nanoemulsion can be used to modulate the release of the drug from tablet matrix. The paper by P. Severino et al. gives an account of potential of solid lipid nanocarriers for the oral delivery of drugs and peptides. The authors have provided information about the lipids that can be used for oral delivery, role of lipids in the oral delivery, toxicological aspects of lipid nanocarriers, and products under clinical development. S. Banerjee et al. have given a complete overview of polyethylene-glycol- (PEG-) based conjugates for drug delivery. The contribution fosters understanding design aspects of and chemistry behind PEG-based nano-architectures for drug delivery. Furthermore, the paper has a detailed discussion on the various PEG-conjugates available in the pharmaceutical market. Contribution by A. Garcia et al. highlights the potential of particle replication in nonwetting templates (PRINT), a platform technology based on lithographic techniques for drug delivery applications. The contribution clearly demonstrates potential of PRINT technology to generate particles of various, but precise, morphology for a variety of drugs and biotechnology-based therapeutics (proteins and siRNA). The application of PRINT technology for generating aerosols for pulmonary applications has also been described. This contribution is an example of the attributes required from a nanofabrication technique to circumvent manufacturing-related issues in pharmaceutical nanotechnology. The paper by F. Lallemand et al. delineates various aspects of and challenges in ocular drug delivery and systematically describes development of Novasorb, a cationic nanoemulsion-based platform ocular delivery system. The authors have furnished a detailed description of the formulation development aspects and ocular safety of excipients which is followed by in vivo proof-of-concept and clinical development. This paper gives clear insight into various challenges faced for developing nanomedicine for ocular delivery. The paper by J. D. Heidel and T. Schleup throws light on the various applications of self-assembled nanocarriers consisting of cyclodextrin based polymers. The authors describe various developmental aspects of two platforms based on cyclodextrin-based polymers (Cyclosert and RONDEL) which can enable efficient delivery of drugs or nucleic-acid-based therapeutics. The translational aspects of both the nanocarriers and in vivo proof-of-concept have also been furnished in this paper. The research paper by J. Rios-Doria et al. gives insight into developmental aspects of a pH sensitive cross-linked polymeric micelle technology (IVECT). The authors describe synthesis of the polymeric micelles, their ability to encapsulate various drugs, and in vivo proof-of-concept for anticancer drugs like daunorubicin and BB4007431. In a nutshell, we believe that this special issue would give readers insight into various aspects involved in translating nanotechnology from bench to pharmaceutical market. Moreover, the special issue also includes some contributions about nanotechnologies that are currently under clinical development. Abhijit A. Date Rajesh R. Patil Riccardo Panicucci Eliana B. Souto Robert W. Lee

Preclinical study of the cyclodextrin-polymer conjugate of camptothecin CRLX101 for the treatment of gastric cancer

Camptothecin showed remarkable anticancer activity in animal models of cancer but was restricted in clinical use for its adverse toxicity in patients. The preclinical efficacy of CRLX101, a nanoparticle (NP) assembly containing cyclodextrin-based polymer and camptothecin was evaluated by in vitro cytotoxicity in gastric cancer cell lines and in vivo antitumor effects in human gastric cancer cell line BGC823 xenografts. Treated tumor sections were analyzed for presence of NPs and compared with vehicle control tumors for hypoxia and angiogenesis. Gastric cancer cell lines showed high in vitro cytotoxicity for CRLX101 and also showed strong antitumor activity in vivo. Electron micrographs revealed the intracellular presence of NPs in close proximity to vesicles. A significant decrease in expressions of carbonic anhydrase, VEGF, and CD31 proteins in treated tumors indicated an inhibition of hypoxia and angiogenesis. The results provide preclinical data for gastric adenocarcinoma. From the Clinical EditorThis study describes a nanoparticle assembly containing cyclodextrin-based polymer and camptothecin, resulting in increased bioavailability of camptothecin, an effective but toxic anti-cancer agent. The antitumor effects and safety profile were demonstrated in a gastric carcinoma cell line.

Adsorption mechanism-based screening of cyclodextrin polymers for adsorption and separation of pesticides from water

A rational screening of cyclodextrin-based polymer (CDP), in terms of the relationship between adsorption potential and adsorbent-adsorbate, was investigated to adsorb and separate pesticides from water. Seven spherical porous CDPs were prepared with onefold or composite cyclodextrin(s) as complex and epichlorohydrin as cross-linking reagent. The adsorption kinetics and isotherms of the polymers toward a mixture of ten distinct pesticides clearly demonstrate that the adsorbents with a homogeneous open network structure can absorb pesticides via multiple adsorption interactions such as CD inclusion, loading into swelling water and physical adsorption on network. The multivariate regression analysis distinguishes the quantitative contributions of polymer properties to its adsorption potential, among which CD content, swelling capacity and pore size appear to be major influencing factors. Consequently, a facile mixture of three CDPs (i.e., β-CDP, RM-CDP and HP-CDP) was screened to obtain above prerequisite properties. The multiplex polymer could superiorly separate the pesticides at environmentally relevant levels from water.

Systemic delivery of siRNA via targeted nanoparticles in patients with cancer: Results from a first-in-class phase I clinical trial.

3022 Background: Systemically delivered small interfering RNA (siRNA) would allow targeting oncogenic molecules beyond current approaches. We report on the first siRNA trial with a targeted nanoparticle delivery system. Methods: Open-label, dose- escalation trial in pts with solid refractory cancers receiving 4 i.v. infusions (30 min) on d 1, 3, 8, and 10 of 21-d cycles. CALAA-01 nanoparticles consisted of a cyclodextrin-based polymer, transferrin protein (hTf) targeting ligand, polyethylene glycol (PEG) for stability, and siRNA against ribonucleotide reductase M2 (RRM2). The 70 nm particles were designed to minimize renal clearance and allow tumor vasculature permeation with binding to tumor hTf receptors (TfR). Primary endpoints (safety, MTD determination) were based on the first cycle. Results: 15 pts accrued to 5 dose levels (3, 9, 18, 24, 30 mg/m2), median age 62 (range 53-85). Most common histologies: GI (4), melanoma (3). Dose escalation progressed with no DLTs. Most common treatment-related AEs: fatigue (47%), fever/chills (33%), allergic (33%), constipation (33%), nausea/vomiting (20%), all g1-2. 1 pt had g3 anemia and 2 pts had g2 thrombocytopenia, all shortly after CALAA-01 infusions with rapid recovery. 1 pt had possibly-related sinus bradycardia (g2). No objective tumor responses were seen; 1 pt at highest dose had stable metastatic melanoma for 4 mo, a change from prior course. Biopsies in 3 pts (melanoma) showed particles in tumors. At 30 mg/m2 RRM2 knockdown (mRNA and protein) was seen with confirmation of mechanism by specific cleavage sequence (RACE-PCR). TfR was not downregulated in cancer cells. Conclusions: Systemic delivery of siRNA via targeted nanoparticles is safe and can induce specific, siRNA-mediated gene silencing. This approach could be expanded to any currently undruggable cancer therapy target. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Calando Calando Calando Calando

Synthesis and evaluation of cyclodextrin-based polymers for patulin extraction from aqueous solutions

Patulin is a mycotoxin produced by fungi that contaminate fruits, juices, and other agricultural commodities. Sorption properties of polyurethane-beta-cyclodextrin polymers were evaluated for the ability to remove patulin from solutions, including apple juice. Freundlich isotherm analysis determined the polymers possess a degree of heterogeneity. Evaluation of the polymers by solid phase extraction analysis indicated patulin sorption is enhanced in aqueous environments. Polymers crosslinked with tolylene 2,4-diisocyanate were suitable for extraction of patulin from apple juice. Quantum chemical studies of the interactions of patulin and beta-cylcodextrin using the PM3 semi-empirical method infer patulin is capable of binding to the polymer in multiple modes. Certain of these bound complexes possess intermolecular hydrogen bond interactions between the primary hydroxyls of beta-cyclodextrin and patulin. These nanoporous cyclodextrin polymers exhibit favorable properties to assist the detection of patulin in aqueous solutions.

Preclinical Results of Camptothecin-Polymer Conjugate (IT-101) in Multiple Human Lymphoma Xenograft Models

Camptothecin (CPT) has potent broad-spectrum antitumor activity by inhibiting type I DNA topoisomerase (DNA topo I). It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with relapsed/refractory lymphoma, but with only modest activity. Recently, we have developed a cyclodextrin-based polymer conjugate of 20-(S)-CPT (IT-101). In this study, we evaluated the preclinical antilymphoma efficacy of IT-101 as compared with CPT-11. We determined an in vitro cytotoxicity of IT-101, CPT-11, and their metabolites against multiple human lymphoma cell lines. In human lymphoma xenografts, the pharmacokinetics, inhibitions of tumor DNA topo I catalytic activity, and antilymphoma activities of these compounds were evaluated. IT-101 and CPT had very high in vitro cytotoxicity against all lymphoma cell lines tested. As compared with CPT-11 and SN-38, IT-101 and CPT had longer release kinetics and significantly inhibit higher tumor DNA topo I catalytic activities. Furthermore, IT-101 showed significantly prolonged the survival of animals bearing s.c. and disseminated human xenografts when compared with CPT-11 at its maximum tolerated dose in mice. The promising present results provide the basis for a phase I clinical trial in patients with relapsed/refractory lymphoma.

Release of a Poorly Soluble Drug from Cross-Linked β-Cyclodextrin-Based Polymer Pellets Prepared via Extrusion/Spheronisation

The aim of this study was to evaluate the use of a β-cyclodextrin-based polymer, and more exactly a highly crosslinked cyclodextrin-based polyester, as an excipient to accelerate the release of poorly soluble drugs from pellets obtained by extrusion-spheronisation. The cross-linked β-cyclodextrin based polymer was associated to microcrystalline cellulose to facilitate the process of extrusion/spheronisation, and nimesulide was chosen as a poorly soluble model drug. Different formulations with natural α and β-cyclodextrins, or a β-cyclodextrin-based polymer associated to microcrystalline cellulose and 10 % (w/w) of nimesulide were compared. Yield between 800 and 1000 μm, pellets dimensions and surface morphology by scanning electron microscopy (SEM), Fourier Transform - Infrared Spectroscopy (FT-IR) spectra, disintegration and in-vitro drug release were evaluated. Due to pellet disintegration, accelerated dissolution of nimesulide was achieved with β-cyclodextrin-based polymer, i.e. 75±2% in 120 min vs. 12±1% for pellets with only microcrystalline cellulose (p < 0.05). On the other hand, pellets with the same percentage of cyclodextrins as cyclodextrinbased polymer, were not disintegrated, and the dissolution was limited around 35±1% after 120 min, with a release linked to the porosity enhancement by leaching of water soluble excipients. In conclusion, the formulations of nimesulide with cross-linked β-cyclodextrin-based polymer appears the most promising for testing in vivo bioavailability studies.

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C. Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.

A dose-finding pharmacokinetic study of IT-101, the first de novo designed nanoparticle therapeutic, in refractory solid tumors

Background: IT-101 is the first de novo designed experimental nanoparticle therapeutic for cancer and is comprised of a cyclodextrin-based polymer conjugate of camptothecin (CPT) that self-assembles into nanoparticles with diameter of ca. 40 nm and near neutral surface charge. The nanoparticle properties have been designed to accomplish extended circulation, tumor penetration, and slow intracellular drug release to enhance the mechanism of action of the CPT. Methods: Patients with refractory solid tumors received IT-101 by 90 minute IV infusions on days 1, 8, and 15 of a 28 day cycle over a dose range of 6 mg/m^2/wk to 18 mg/m^2/wk. Dose escalation was conducted with a Simon’s design. Dose limiting toxicity (DLT) was determined over the first cycle. Progressive disease (PD) was defined by RECIST (CT) results. Pharmacokinetics of free and total CPT in blood and urine were collected over after the 1st dose. Patients completing 6 cycles with stable disease (SD) or better were eligible to receive compassionate IT-101 at the same dose level every other week (QOW). Results: Twelve patients [6 (6 mg/m^2), 3 (12 mg/m^2), and 3 (18 mg/m^2)] were enrolled. 4 of 12 (33%) had PD by CT. 8 of 12 (66%) did not have PD. (6-SD and 2 clinical deterioration (CD). Hematologic toxicity was the DLT. The MTD was 2 cycles, SD was observed at cumulative doses of 18 mg/m^2/mo to 27 mg/m^2/mo. Three patients (pancreatic, renal cell, NSCLC) completed 6 cycles and received treatment QOW with a PFS of ≥18, 14, and 11 months, respectively. Over 7 days, the mean plasma total CPT and free CPT t_(½ β) were 39 ± 1.1 and 51 ± 1.5 hrs, respectively. The AUC of free and total CPT were linear with 9.9 ± 4.0 % of the CPT dose free. Urinary excretion averaged 16 ± 9.1% of the total CPT dose in the first 48 hrs. Conclusions: These data appear to demonstrate that IT-101 is safe, tolerable, and is associated with acceptable toxicity at the MTD. SD was maintained in patients treated beyond 6 cycles QOW at doses of 6 to 9 mg/m^2/wk. In addition, twice monthly regimens maintained SD with minimal drug-related toxicity. A QOW schedule (allowing for 99% elimination of CPT) is warranted in future clinical trial.

Preclinical Results of the Camptothecin-Polymer Conjugate IT-101 in Multiple Human Lymphoma Xenografts.

Camptothecin (CPT), a natural alkaloid isolated from Camptotheca acuminata, has potent broad spectrum antitumor activity by inhibiting type I DNA topoisomerase. It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with refractory/relapsed lymphoma, but with only modest activity. For intravenous administration, we have developed IT-101, a ca. 40 nm diameter nanoparticle that is an assembly of cyclodextrin-based polymer conjugates of 20-(S)-CPT. The purpose of this study is to compare the preclinical efficacy of IT-101 with CPT-11 both in vitro and in vivo. Fluorescence microscopy studies demonstrated that incubation of a human lymphoma cell line with IT-101 resulted in the uptake and intracellular accumulation of CPT. Based on the demonstration of in vitro cytotoxity of IT-101 against multiple human lymphoma cell lines, we initiated experiments in xenograft models of lymphoma. In subcutaneous human xenograft models, a single cycle of three weekly doses of intravenous IT-101 at 10 mg/kg (CPT equivalents) showed significantly potent antitumor activity against Daudi, Karpas 299, and L540 cell lines compared to three weekly doses of intraperitoneal CPT-11 at its maximum tolerated dose in mouse of 100 mg/kg (P < 0.0001, P = 0.0072, and P < 0.0001, respectively). In the same animal models, IT-101 led to pathologically complete remissions in 78%, 44%, and 78% of animals inoculated with Daudi, Karpas 299, and L540 cell lines, respectively. In disseminated human xenograft models, IT-101, administered using the same dosing schedule, significantly prolonged the survival of animals intravenously injected with either Daudi or Karpas 299 cell lines when compared to CPT-11 (P < 0.0001 and P = 0.0049, respectively). The promising present results in a variety of lymphomas provide the basis for a phase I/II clinical trial in patients with refractory/relapsed lymphoma.

Highly crosslinked ionic β-cyclodextrin polymers and their interaction with heavy metals

A new aqueous insoluble ionic β-cyclodextrin polymer (PYR) has been synthesized and a potentiometric study of the binary Cu(II)-PYR system is performed to calculate the complexation constants (as logβ in heterogeneous medium). The mathematical processing of the pH-metric data gave the formation constants of Cu(II) complexes and the related species distributions. The model is compatible with the presence of five complex species in the range of pH 2.5–7. Stoichiometry indicates the probable involvement of the alcoholate functionalities of the ligand in the complexation. The capacity of the polymer with respect to metal ions retention is evaluated for both Cu(II) and Cd(II) (chosen as target probes). The possibility to recover the sorbed Cd(II) is also tested by using acidic pH solutions. A complete recovery is obtained and the stability of the polymer is verified over ten steps of retention and desorption. To understand the complexation mechanism involved, two other cyclodextrin-based polymers are synthesized which are characterized by the presence of naphthalic dicarboxylic and carbonate groups as spacers. Their interactions with Cu(II) or Cd(II) are studied. Since the β-cyclodextrin polycarbonate polymer does not have acidic groups on the spacer, it is interesting to compare metal ions retention between this material, which does not present a real cation exchange site, and PYR.

Preclinical Efficacy of Campthothecin-Polymer Conjugate (IT-101) in Human Burkitt Lymphoma Bearing Mice.

First line combination chemotherapy can result in long-term disease-free survival (DFS) in 65–90% of patients with Burkitt lymphoma (BL) depending on prognostic factors at presentation. However, only a small number of patients with relapsed/refractory diseases can achieve long-term DFS after the high-dose therapy and hematopoietic stem cell rescue. Irinotecan (CPT-11) and topotecan, two analogues of campthothecin (CPT), have been successfully used alone or in combination with other cytotoxic agents as salvage treatments for patients with relapsed/refractory non-Hodgkin's lymphoma (NHL) other than BL. A disadvantage of CPT-11 and topotecan observed in early clinical trials was a high incidence of treatment-related toxicity (TRT). IT-101, a nanoparticulate conjugate of 20(S)-camptothecin and a cyclodextrin-based polymer, has recently been developed to minimize TRT and increase anti-tumor activity. To evaluate preclinical efficacy and toxicity of IT-101, we conducted an in vivo study in nude mice bearing subcutaneous xenografts from human BL-derived Daudi cell line stably transfected with plasmid DNA containing firefly luciferase (ffluc) reporter gene. The tumor-derived ffluc activity was evaluated in vivo using an IVIS 100 imaging system (Xenogen); background ffluc luminescence was defined as ≤106 p/sec/cm2/sr. Ten days after subcutaneous tumor injection, 42 nude mice with tumor-derived ffluc activity of >108 p/sec/cm2/sr were allocated into six different treatment groups as followings: Group 1 (no therapy); Group 2 (CPT-11 100mg/kg, ip, qwk × 3); Group 3 (IT-101 5mg/kg, iv, qwk × 3); Group 4 (IT-101 10mg/kg, iv, qwk × 3); Group 5 (IT-101 15mg/kg, iv, qwk × 2); and Group 6 (IT-101 single dose of 15mg/kg, iv). The average tumor-derived ffluc activity among all of the treatment groups was equally distributed. Tumor burden was monitored weekly by tumor-derived ffluc activity and physical tumor measurements. After completion of the treatment, there were two treatment-related deaths (TRDs; body weight loss >20%) and one non-TRD in group 5 and 6, respectively. At the 3rd week after starting therapy, all treatment groups that received two or three weekly doses of IT-101 (Groups 3, 4, and 5) had 100% complete tumor regression (CTR), determined by ffluc activity (<106 p/sec/cm2/sr) and remained tumor free at 90 days post-therapy. In contrast, 57% (4/7) of mice in Group 2 and 83% (5/6) of mice in Group 6 had initial CTR but all had tumor re-growth by day 69. These results demonstrate that a single dose of IT-101 at 15mg/kg is not sufficiently efficacious while two weekly doses of IT-101 at 15mg/kg are associated with unacceptable toxicity. However, we are able to demonstrate that the three weekly doses of IT-101 at 5 or 10mg/kg are well tolerated and can produce superior anti-tumor activity against Daudi cell line as compared with CPT-11.

Preclinical Efficacy of the Camptothecin-Polymer Conjugate IT-101 in Multiple Cancer Models

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 LS174T colon carcinoma xenografts are elucidated. All multiple dosing schedules administered over 15 to 19 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 non-small-cell lung cancer, H69 small-cell lung cancer, Panc-1 pancreatic 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-101 resulted 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-101 is 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.

Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles

Short nucleic acid sequences specific to oncogene targets such as bcl-2, bcr-abl, and c-myc have been shown to exhibit specific anti-cancer activity in vitro through antigene or antisense activity. Efficient in vivo delivery of oligonucleotides remains a major limitation for the therapeutic application of these molecules. We report herein on the preparation of transferrin-modified nanoparticles containing DNAzymes (short catalytic single-stranded DNA molecules) for tumor targeting as well as their biodistribution using various methods of administration in the mouse. Linear, ?- cyclodextrin-based polymers are complexed with DNAyzme molecules to form sub- 50 nm particles termed “polyplexes”. The surface properties of the cyclodextrincontaining polyplexes are modified by exploiting the ability of the ?-cyclodextrin substructure and adamantane to form inclusion complexes. Accordingly, conjugates of adamantane with poly(ethylene glycol) (PEG) are prepared and combined with the polyplexes. The adamantane form inclusion complexes with the surface cyclodextrins of the polyplexes to provide a sterically stabilizing layer of PEG. The stabilized polyplexes are also modified with transferrin for increasing targeting to tumor cells expressing transferrin receptors. The preparation, characterization, and in vitro application of these nanoparticles are discussed. The transferrin-polyplexes containing fluorescently-labeled DNAzyme molecules are administered to tumorbearing nude mice and their biodistribution and clearance kinetics are monitored using a fluorescence imaging system. Four methods of administration are studied: intraperitoneal bolus and infusion, intravenous bolus, and subcutaneous injection. DNAzymes packaged in polyplex formulations are concentrated and retained in tumor tissue and other organs, whereas unformulated DNAzyme is eliminated from the body within 24 hours post-injection. Intravenous and intraperitoneal bolus injections result in the highest fluorescent signal (DNAzyme) at the tumor site. Tumor cell uptake is observed with intravenous bolus injection only, and intracellular delivery requires transferrin targeting.