Phosphorylcholine-Grafted Molecular Bottlebrush-Doxorubicin Conjugates: High Structural Stability, Long Circulation in Blood, and Efficient Anticancer Activity.

Controlling the particle structure of tumor-targeting nanomedicines in vivo remains challenging but must be achieved to control their in vivo fate and functions. Molecular bottlebrushes (MBs), where brush side chains are densely grafted from a main chain, have recently received attention as building blocks of polymer-based prodrugs because their rigid structure would be expected to demonstrate high structural stability in vivo. Here, we synthesized a poly(methacryloyloxyethyl phosphorylcholine) (pMPC)-grafted molecular bottlebrush (PCMB) conjugated with a cancer drug, doxorubicin (DOX), via an acid-cleavable hydrazone bond. A pMPC-based linear polymer (LP) conjugated with DOX was also prepared for comparison. We confirmed the lack of structural transition in the PCMB between before and after conjugation with DOX using small-angle light and X-ray scattering techniques, whereas the structure of LP was significantly influenced by DOX conjugation and transformed from a random-coil structure to a large agglomerate via hydrophobic interactions among DOXs. Although PCMB-DOX and LP-DOX showed comparable tissue permeability, pharmacokinetics, and ability to accumulate in tumor tissues, the antitumor efficacy of PCMB-DOX was better than that of LP-DOX. This was presumably due to the formation of LP-DOX agglomerates. The diffusion of cleaved DOX would be restricted in the hydrophobic core of the agglomerate, resulting in the DOX release at the tumor site being compromised. In contrast to LP-DOX, DOX release from PCMB-DOX was not compromised after accumulation in tumor tissues because it did not form such an agglomerate, resulting in the strong antitumor effect. We have demonstrated the potential of MBs as building blocks of drug carriers and believe that these findings can contribute to the design of polymer-based nanomedicines.

Development of potent antipseudomonal ß-lactams by means of polycarboxylation of aminopenicillins.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that presents a serious risk to immunosuppressed individuals and other extremely vulnerable patients such as those in intensive care units. The emergence of multidrug-resistant Pseudomonas strains has increased the need for new antipseudomonal agents. In this study, a series of amino group-modified aminopenicillin derivatives was synthesized that have different numbers of carboxyl groups and structurally resemble carboxypenicillin-ureidopenicillin hybrids, and their antipseudomonal activities were evaluated. Among the derivatives synthesized, diethylenetriaminepentaacetic acid (DTPA)-modified amoxicillin (DTPA-Amox) showed potent antipseudomonal activity, not only against the laboratory strain PAO1 but also against clinically isolated Pseudomonas strains that were resistant to piperacillin and carbenicillin. DTPA-Amox had no obvious cytotoxic effects on cultured mammalian cells. In addition, in an in vivo model of leukopenia, DTPA-Amox treatment produced a moderate but statistically significant improvement in the survival of mice with P. aeruginosa strain PAO1 infection. These data suggest that polycarboxylation by DTPA conjugation is an effective approach to enhance antipseudomonal activity of aminopenicillins.

Singlet oxygen phosphorescence detection in vivo identifies PDT-induced anoxia in solid tumors.

Real-time surveillance of photodynamic therapy (PDT) has been desired by the research community for a long time. The impact of the treatment is encoded in the phosphorescence kinetics of its main mediator: singlet oxygen. We report successful in vivo measurements of these weak kinetics through the skin of living mice after systemic drug application. Using special high transmission optics centered around 1200, 1270 and 1340 nm, singlet oxygen phosphorescence can be clearly discriminated from other signals. N-(2-Hydroxypropyl)methacrylamide copolymers conjugated with pyropheophorbide-a exhibit highly selective accumulation in tumors. Signals of this drug in tumors were compared to those in normal tissue. In both places, the major part of the signal could be identified as arising from drug still circulating in the bloodstream. Despite high concentrations of extravasated drug in the tumors due to the EPR effect, nearly no signal could be detected from these photosensitizers in vivo, contradicting in vitro experiments. We propose that the reason for this discrepancy is oxygen depletion in tumor tissue in vivo, even at moderate (at PDT scale) illumination intensities, soon after the start of the illumination. These results underline the importance of singlet oxygen surveillance during PDT treatment.

Synthesis of Pegylated Manganese Protoporphyrin as a Catalase Mimic and Its Therapeutic Application to Acetaminophen-Induced Acute Liver Failure.

Metalloporphyrin derivatives have been investigated for their therapeutic potential for oxidative stress-related diseases because of their scavenging of reactive oxygen species (ROS). Here, we describe the synthesis, physicochemical properties, and ROS-scavenging activities of one such derivative-polyethylene glycol (PEG)-conjugated manganese protoporphyrin (PEG-MnPP). Carboxyl groups of the protoporphyrin ring at the C6 and C7 positions were first conjugated with ethylenediamine to introduce amino groups into the protoporphyrin structure. The amino groups were then reacted with succinimidyl PEG, with an average molecular weight of 2000, to obtain pegylated protoporphyrin (PEG-PP). Manganese was chelated to the protoporphyrin ring by incubating PEG-PP and manganese acetate in methanol. Dynamic light scattering and fluorescent spectrometry analyses revealed that PEG-MnPP self-assembled into nanoparticles in aqueous media with an apparent diameter of 70 nm. PEG-MnPP effectively eliminated hydrogen peroxide from cell culture media and protected cultured mammalian cells from toxic insults induced by hydrogen peroxide exposure or by 6-hydroxydopamine treatment. Intravenous administration of PEG-MnPP to mice significantly suppressed acute liver failure that had been induced by acetaminophen overdose. These data warrant additional investigation to study the therapeutic potential of PEG-MnPP as a water-soluble metalloporphyrin-based catalase mimic for oxidative stress-associated diseases.

Enhanced Permeability and Retention Effect as a Ubiquitous and Epoch-Making Phenomenon for the Selective Drug Targeting of Solid Tumors.

In 1979, development of the first polymer drug SMANCS [styrene-co-maleic acid (SMA) copolymer conjugated to neocarzinostatin (NCS)] by Maeda and colleagues was a breakthrough in the cancer field. When SMANCS was administered to mice, drug accumulation in tumors was markedly increased compared with accumulation of the parental drug NCS. This momentous result led to discovery of the enhanced permeability and retention effect (EPR effect) in 1986. Later, the EPR effect became known worldwide, especially in nanomedicine, and is still believed to be a universal mechanism for tumor-selective accumulation of nanomedicines. Some research groups recently characterized the EPR effect as a controversial concept and stated that it has not been fully demonstrated in clinical settings, but this erroneous belief is due to non-standard drug design and use of inappropriate tumor models in investigations. Many research groups recently provided solid evidence of the EPR effect in human cancers (e.g., renal and breast), with significant diversity and heterogeneity in various patients. In this review, we focus on the dynamics of the EPR effect and restoring tumor blood flow by using EPR effect enhancers. We also discuss new applications of EPR-based nanomedicine in boron neutron capture therapy and photodynamic therapy for solid tumors.

Structural Determination of the Nanocomplex of Borate with Styrene-Maleic Acid Copolymer-Conjugated Glucosamine Used as a Multifunctional Anticancer Drug.

The development of effective anticancer drugs is essential for chemotherapy that specifically targets cancer tissues. We recently synthesized a multifunctional water-soluble anticancer polymer drug consisting of styrene-maleic acid copolymer (SMA) conjugated with glucosamine and boric acid (BA) (SGB complex). It demonstrated about 10 times higher tumor-selective accumulation compared with accumulation in normal tissues because of the enhanced permeability and retention effect, and it inhibited tumor growth via glycolysis inhibition, mitochondrial damage, and thermal neutron irradiation. Gaining insight into the anticancer effects of this SGB complex requires a determination of its structure. We therefore investigated the chemical structure of the SGB complex by means of nuclear magnetic resonance, infrared (IR) spectroscopy, and liquid chromatography-mass spectrometry. To establish the chemical structure of the SGB complex, we synthesized a simple model compound─maleic acid-glucosamine (MAG) conjugate─by using a maleic anhydride (MA) monomer unit instead of the SMA polymer. We obtained two MAG-BA complexes (MAGB) with molecular weights of 325 and 343 after the MAG reaction with BA. We confirmed, by using IR spectroscopy, that MAGB formed a stable complex via an amide bond between MA and glucosamine and that BA bound to glucosamine via a diol bond. As a result of this chemical design, identified via analysis of MAGB, the SGB complex can release BA and demonstrate toxicity to cancer cells through inhibition of lactate secretion in mild hypoxia that mimics the tumor microenvironment. For clinical application of the SGB complex, we confirmed that this complex is stable in the presence of serum. These findings confirm that our design of the SGB complex has various advantages in targeting solid cancers and exerting therapeutic effects when combined with neutron irradiation.

ATP exposure stimulates glutathione efflux as a necessary switch for NLRP3 inflammasome activation.

The NLRP3 inflammasome is a multiprotein complex responsible for the maturation of precursor forms of interleukin (IL)-1ß and IL-18 into active proinflammatory cytokines. Increasing evidence suggests that modulation of redox homeostasis contributes to the activation of the NLRP3 inflammasome. However, specific mechanistic details remain unclear. We demonstrate here that ATP exposure evoked a sharp decrease in glutathione (GSH) levels in macrophages, which led to NLRP3 inflammasome activation. We detected an increase in GSH levels in culture supernatants that was comparable to the GSH decrease in macrophages, which suggests that exposure to ATP stimulated GSH efflux. Exogenous addition of P2X7 receptor antagonist, GSH, or the oxidized form GSSG attenuated this efflux. Also, exogenous GSH or GSSG strongly inhibited NLRP3 inflammasome activation in vitro and in vivo. These data suggest that GSH efflux controls NLRP3 inflammasome activation, which may lead to development of novel therapeutic strategies for NLRP3 inflammasome-associated disorders.

EPR-Effect Enhancers Strongly Potentiate Tumor-Targeted Delivery of Nanomedicines to Advanced Cancers: Further Extension to Enhancement of the Therapeutic Effect.

For more than three decades, enhanced permeability and retention (EPR)-effect-based nanomedicines have received considerable attention for tumor-selective treatment of solid tumors. However, treatment of advanced cancers remains a huge challenge in clinical situations because of occluded or embolized tumor blood vessels, which lead to so-called heterogeneity of the EPR effect. We previously developed a method to restore impaired blood flow in blood vessels by using nitric oxide donors and other agents called EPR-effect enhancers. Here, we show that two novel EPR-effect enhancers-isosorbide dinitrate (ISDN, Nitrol®) and sildenafil citrate-strongly potentiated delivery of three macromolecular drugs to tumors: a complex of poly(styrene-co-maleic acid) (SMA) and cisplatin, named Smaplatin® (chemotherapy); poly(N-(2-hydroxypropyl)methacrylamide) polymer-conjugated zinc protoporphyrin (photodynamic therapy and imaging); and SMA glucosamine-conjugated boric acid complex (boron neutron capture therapy). We tested these nanodrugs in mice with advanced C26 tumors. When these nanomedicines were administered together with ISDN or sildenafil, tumor delivery and thus positive therapeutic results increased two- to four-fold in tumors with diameters of 15 mm or more. These results confirmed the rationale for using EPR-effect enhancers to restore tumor blood flow. In conclusion, all EPR-effect enhancers tested showed great potential for application in cancer therapy.

Unraveling the role of Intralipid in suppressing off-target delivery and augmenting the therapeutic effects of anticancer nanomedicines.

Intralipid, a clinically used lipid emulsion, was reportedly utilized as one strategy to suppress off-target delivery of anticancer nanomedicines; Intralipid also effectively improved drug delivery to tumors and produced better therapeutic effects. However, the mechanisms involved-the why and how-in Intralipid's facilitation of delivery of nanomedicines to tumors have not yet been reported in detail. In this study, we investigated Intralipid and discovered the beneficial effects of Intralipid pretreatment when using three anticancer nanomedicines, including the clinically approved drug doxorubicin (Doxil). Intralipid pretreatment induced a 40% reduction in liver uptake of a polymeric nanoprobe used in photodynamic therapy as well as a 1.5-fold-increased nanomedicine accumulation in tumors. This increased accumulation consequently led to significantly better therapeutic effects, and this finding was validated by using Doxil. As an interesting result, Intralipid pretreatment significantly prolonged the plasma half-life of nanomedicines in normal healthy mice but not in tumor-bearing mice, which suggests that tumors become an alternative route of nanomedicine delivery when liver delivery is suppressed. Also, we found markedly increased tumor blood flow, as measured by fluorescence angiography, and significantly lower blood viscosity after Intralipid pretreatment. All our results together indicate that Intralipid treatment not only suppressed off-target nanomedicine delivery by the reticuloendothelial system, but more important, it enhanced nanomedicine delivery to tumors by improving tumor blood flow, which is key to satisfactory drug delivery via the enhanced permeability and retention effect. Significantly better therapeutic outcomes were thus achieved by the strategy of combining utilization of nanomedicines and Intralipid pretreatment. STATEMENT OF SIGNIFICANCE: Off-target delivery to organs such as the liver and obstructed tumor blood flow as is often seen in advanced cancers are major barriers to the therapeutic efficacy of anticancer nanomedicines. Intralipid has been shown effective for suppressing nanomedicine accumulation in the liver, resulting in improved anticancer effects. Unraveling the mechanisms involved in this process will be greatly helpful for the clinical application of anticancer nanomedicines. We reported here that Intralipid could also significantly increase tumor delivery of nanomedicine, which is beneficial for improving tumor blood flow and lowering blood viscosity. To our knowledge, this is the first study to investigate the role of Intralipid in this regard. This knowledge provides a solid rationale for the use of Intralipid in combination with anticancer nanomedicines.

Polymer-conjugated glucosamine complexed with boric acid shows tumor-selective accumulation and simultaneous inhibition of glycolysis.

We synthesized unique water-soluble synthetic-polymer, styrene-maleic acid copolymer (SMA) conjugated glucosamine (SG); which formed a stable complex with boric acid (BA). This complex had a mean particle size of 15 nm by light scattering, and single peak in gel permeation chromatography. The particles were taken up by tumor cells five times faster than free BA in vitro and liberated BA at acidic tumor pH (5-7). Liberated BA inhibited glycolysis and resulted in tumor suppression in vivo. Intravenously injected SGB-complex did bind with albumin, and plasma half-life was about 8 h in mice, and accumulated to tumor tissues about 10 times more than in normal organs. IC50 of SGB-complex for HeLa cells under pO2 of 6-9% was about 20 µg/ml (free BA equivalent), 150 times more potent than free BA. Neutron irradiation of human oral cancer cells with SGB-complex resulted in 16 times greater cell-killing than that without SGB-complex. In vivo antitumor effect was evaluated after neutron irradiation only once in SCC VII tumor bearing mice and significant tumor suppression was confirmed. These results indicate that SGB-complex is a unique multifunctional anticancer agent with much more potent activity under low pO2 conditions as in large advanced cancers.

Exploiting the dynamics of the EPR effect and strategies to improve the therapeutic effects of nanomedicines by using EPR effect enhancers.

The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors that is related to their particular anatomical and pathophysiological characteristics, e.g. defective vascular architecture; large gaps between endothelial cells in blood vessels; abundant vascular mediators such as bradykinin, nitric oxide, carbon monoxide, and vascular endothelial growth factor; and impaired lymphatic recovery. These features lead to tumor tissues showing considerable extravasation of plasma components and nanomedicines. These data comprise the basic theory underlying the development of macromolecular agents or nanomedicines. The EPR effect is not necessarily valid for all solid tumors, because tumor blood flow and vascular permeability vary greatly. Tumor blood flow is frequently obstructed as tumor size increases, as often seen clinically; early stage, small tumors show a more uniform EPR effect, whereas advanced large tumor show heterogeneity in EPR effect. Accordingly, it would be very important to apply enhancers of EPR effect in clinical setting to make EPR effect more uniform. In this review, we discuss the EPR effect: its history, factors involved, and dynamics and heterogeneity. Strategies to overcome the EPR effect's heterogeneity may guarantee better therapeutic outcomes of drug delivery to advanced cancers.

Enhanced Cellular Polysulfides Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin Shock.

Cysteine persulfide and cysteine polysulfides are cysteine derivatives having sulfane sulfur atoms bound to cysteine thiol. Accumulating evidence has suggested that cysteine persulfides/polysulfides are abundant in prokaryotes and eukaryotes and play important roles in diverse biological processes such as antioxidant host defense and redox-dependent signal transduction. Here, we show that enhancement of cellular polysulfides by using polysulfide donors developed in this study resulted in marked inhibition of lipopolysaccharide (LPS)-initiated macrophage activation. Polysulfide donor treatment strongly suppressed LPS-induced pro-inflammatory responses in macrophages by inhibiting Toll-like receptor 4 (TLR4) signaling. Other TLR signaling stimulants-including zymosan A-TLR2 and poly(I:C)-TLR3-were also significantly suppressed by polysulfur donor treatment. Administration of polysulfide donors protected mice from lethal endotoxin shock. These data indicate that cellular polysulfides negatively regulate TLR4-mediated pro-inflammatory signaling and hence constitute a potential target for inflammatory disorders.

Augmentation of EPR Effect and Efficacy of Anticancer Nanomedicine by Carbon Monoxide Generating Agents.

One obstacle to the successful delivery of nanodrugs into solid tumors is the heterogeneity of an enhanced permeability and retention (EPR) effect as a result of occluded or embolized tumor blood vessels. Therefore, the augmentation of the EPR effect is critical for satisfactory anticancer nanomedicine. In this study, we focused on one vascular mediator involved in the EPR effect, carbon monoxide (CO), and utilized two CO generating agents, one is an extrinsic CO donor (SMA/CORM2 micelle) and another is an inducer of endogenous CO generation via heme oxygenase-1 (HO-1) induction that is carried out using pegylated hemin. Both agents generated CO selectively in solid tumors, which resulted in an enhanced EPR effect and a two- to three-folds increased tumor accumulation of nanodrugs. An increase in drug accumulation in the normal tissue did not occur with the treatment of CO generators. In vivo imaging also clearly indicated a more intensified fluorescence of macromolecular nanoprobe in solid tumors when combined with these CO generators. Consequently, the combination of CO generators with anticancer nanodrugs resulted in an increased anticancer effect in the different transplanted solid tumor models. These findings strongly warrant the potential application of these CO generators as EPR enhancers in order to enhance tumor detection and therapy using nanodrugs.

Augmentation of the Enhanced Permeability and Retention Effect with Nitric Oxide-Generating Agents Improves the Therapeutic Effects of Nanomedicines.

Enhanced permeability and retention (EPR) effect-based nanomedicine is a promising strategy for successful anticancer therapy. The EPR effect is based on tumor blood flow. Because advanced large tumors, as frequently seen in clinical settings, are heterogeneous, with regions of defective vasculature and blood flow, achieving the desired tumor drug delivery is difficult. Here, we utilized the EPR effect to increase drug delivery. To augment the EPR effect for improved therapeutic effects of nanomedicine, we exploited vascular mediators-the nitric oxide (NO) generators nitroglycerin (NG), hydroxyurea, and l-arginine. These compounds generate NO in tumors with relatively high selectivity. Using different nanosized drugs in our protocol significantly increased (1.5-2 times) delivery of nanomedicines to different solid tumor models, along with markedly improving (2-3-fold) the antitumor effects of these drugs. Also, in 7,12-dimethylbenz[a]anthracene-induced advanced end-stage breast cancer, often seen in clinical settings, 2 mg/kg polymer-conjugated pirarubicin (P-THP) with NG (0.2 mg/mouse) showed better effects than did 5 mg/kg P-THP, and 5 mg/kg P-THP used with NG resulted in cures or stable tumors (no tumor growth) for up to 120 days. Moreover, in a murine autochthonous azoxymethane/dextran sulfate sodium-induced colon cancer model, NO donors markedly improved the therapeutic effects of P-THP even after just one injection, results that were comparable with those achieved with three weekly P-THP treatments. These findings strongly suggest the potential usefulness of NO donors as EPR effect enhancers to improve the therapeutic efficacy of nanomedicines.

HPMA copolymer conjugate with pirarubicin: In vitro and ex vivo stability and drug release study.

We have developed a tumor environment-responsive polymeric anticancer prodrug containing pirarubicin (THP) conjugated to N-(2-hydroxypropyl) methacrylamide copolymer (PHPMA), [P-THP], through a spacer containing pH-sensitive hydrazone bond, that showed remarkable therapeutic effect against various tumor models and in a human pilot study. Toward clinical development, here we report THP release profile from its HPMA copolymer conjugate, the conjugate stability, protein and cell-binding and solubility of P-THP. Size exclusion chromatography of P-THP (molecular weight 38 kDa) showed similar hydrodynamic volume as bovine serum albumin (BSA) in aqueous solution, with no apparent interactions with BSA, nor aggregation by itself. pH-responsive release of free THP was reconfirmed at pHs 6.5 and lower. The drug release was significantly affected by a type of used buffer. Phosphate buffer seems to facilitate faster hydrazone bond cleavage at pH 7.4 whereas higher stability was achieved in L-arginine solution which yielded only little cleavage and THP release, approx. 15% within 2 weeks at the same pH at 25 °C. Furthermore, ex vivo study using sera of different animal species showed very high stability of P-THP. Incubation with blood showed high stability of P-THP during circulation, without binding to blood cells. These findings revealed that L-arginine solution provides appropriate media for formulation of P-THP infusion solution as tumor-targeted polymeric anticancer drug based on EPR effect.

N-(2-hydroxypropyl)methacrylamide polymer conjugated pyropheophorbide-a, a promising tumor-targeted theranostic probe for photodynamic therapy and imaging.

Tumor-targeted photodynamic therapy (PDT) using polymeric photosensitizers is a promising therapeutic strategy for cancer treatment. In this study, we synthesized a pHPMA conjugated pyropheophorbide-a (P-PyF) as a cancer theranostic agent for PDT and photodynamic diagnostics (PDD). Pyropheophorbide-a has one carboxyl group which was conjugated to pHPMA via amide bond yielding the intended product with high purity. In aqueous solutions, P-PyF showed a mean particle size of ∼200 nm as it forms micelle which exhibited fluorescence quenching and thus very little singlet oxygen (1O2) production. In contrast, upon disruption of micelle strong fluorescence and 1O2 production were observed. In vitro study clearly showed the PDT effect of P-PyF. More potent 1O2 production and PDT effect were observed during irradiation at ∼420 nm, the maximal absorbance of pyropheophorbide-a, than irradiation at longer wavelength (i.e., ∼680 nm), suggesting selection of proper absorption light is essential for successful PDT. In vivo study showed high tumor accumulation of P-PyF compared with most of normal tissues due to the enhanced permeability and retention (EPR) effect, which resulting in superior antitumor effect under irradiation using normal xenon light source of endoscope, and clear tumor imaging profiles even in the metastatic lung cancer at 28 days after administration of P-PyF. On the contrary irradiation using long wavelength (i.e., ∼680 nm), the lowest Q-Band, exhibited remarkable tumor imaging effect with little autofluorescence of background. These findings strongly suggested P-PyF may be a potential candidate-drug for PDT/PDD, particularly using two different wavelength for treatment and detection/imaging, respectively.

An unusual complication of suprapubic catheter insertion.

A patient who had a small bowel mesentery perforation following insertion of a suprapubic catheter (SPC) is described. He had no bowel complaints immediately following the procedure, but presented 10 weeks later with insidious onset bowel obstruction due to the kink caused by the catheter. This complication occurred despite cystoscopy control and adequate bladder distension prior to the procedure. This isolated case illustrates the fact that regardless of the ease and frequency of SPC insertion, complications do occur.

The risk of bladder and autonomic dysfunction in a community cohort of Parkinson's disease patients and normal controls.

A questionnaire investigating bladder problems, symptoms of autonomic dysfunction, social handicap and depression was mailed to a sample of patients with Parkinson's disease (PD) and to elderly control subjects without PD. The patients reported two-fold greater risk of bladder problems and four-fold risk of autonomic problems compared to the controls. Erectile dysfunction was nearly twice as frequent in patients compared to controls. Depressive symptoms in the PD group were predictive of bladder problems and autonomic impairment and also poorer social functioning and dependency in activities of daily living. No associations between bladder and autonomic dysfunction, age, or severity/duration of PD were found. This investigation shows that the risk of bladder, autonomic and erectile dysfunction is significantly greater in patients with PD compared to a control group.