Fabrizio Marcucci

Active targeting with particulate drug carriers in tumor therapy: fundamentals and recent progress

Drug therapy for the treatment of tumors is often limited by a narrow therapeutic index. One approach that overcomes this limitation is the active targeting of tumors with particulate drug carriers. The derivatization of particulate drug carriers with a ligand leads to the selective targeting of the particulate to selected cells, thereby focusing drug delivery. In addition, particulate drug carriers have a high loading capacity, do not need covalent conjugation of the drug and the formulation protects the entrapped drug from enzymatic inactivation. Despite these favorable properties, their therapeutic efficacy in animal models has been reported only in recent years. The use of internalizing ligands and the targeting of intravascular tumor cells and endothelial cells of tumor blood vessels have been instrumental in demonstrating the clinical effectiveness of particulate drug carriers in animal models. As a result, several actively targeted particulate carriers have now entered, or are about to enter, clinical investigation. Recent findings, for example, the identification of cell-penetrating peptides with restricted cell selectivity, suggest that further improvements in this approach are likely in the near future.

Hepatitis viruses and non-Hodgkin lymphoma: epidemiology, mechanisms of tumorigenesis, and therapeutic opportunities

Over the past 2 decades considerable evidence has accumulated on the association between hepatitis C virus (HCV) and hepatitis B virus (HBV) and several hematologic malignancies, most notably B-cell non-Hodgkin lymphoma (NHL). In this review we summarize this evidence, address possible mechanisms whereby hepatitis viruses may contribute to lymphomagenesis, and discuss the therapeutic fallouts from this knowledge. Most of this evidence is on HCV, and this is the main focus of the review. Moreover, we mainly address the association with NHL, the most prevalent hematologic malignancy, and the most extensively investigated with regard to an association with hepatitis viruses. Available evidence on the association with other hematologic malignancies is also addressed briefly.

How to improve exposure of tumor cells to drugs: promoter drugs increase tumor uptake and penetration of effector drugs.

Solid tumors are characterized by an abnormal architecture and composition that limit the uptake and distribution of antitumor drugs. Over the last two decades, drugs have been identified that improve the tumor uptake and distribution of drugs that have direct antitumor effects. We propose to refer to these drugs as promoter drugs, and as effector drugs to drugs that have direct antitumor effects. Some promoter drugs have received regulatory approval, while others are in active clinical development. This review gives an overview of promoter drugs, by classifying them according to their mechanism of action: promoter drugs that modulate tumor blood flow, modify the barrier function of tumor vessels, induce tumor cell killing, and overcome stromal barriers. Eventually, we discuss those that we feel are the main conclusions to be drawn from promoter drug research that has been performed so far, and suggest areas of future investigation to improve the efficacy of promoter drugs in cancer therapy.

Pushing tumor cells towards a malignant phenotype: Stimuli from the microenvironment, intercellular communications and alternative roads

The tumor microenvironment produces different types of stimuli capable of endowing tumor cells with an aggressive behavior that is characterized by increased motility, invasiveness and propensity to metastasize, gain of a tumor‐initiating phenotype, and drug resistance. The following classes of stimuli have been reported to promote such a malignant phenotype: (i) solid‐ or fluid‐induced stress; (ii) altered composition of the extracellular matrix; (iii) hypoxia and low pH; (iv) innate and adaptive immune responses; (v) antitumor drugs. The simultaneous presence of more than one of these stimuli, as likely occurs in vivo, may lead to synergistic interactions in the induction of malignant traits. In many cases, the gain of a malignant phenotype is not the result of a direct effect of the stimuli on tumor cells but, rather, a stimulus‐promoted cross‐talk between tumor cells and other cell types within the tumor microenvironment. This cross‐talk is mainly mediated by two classes of molecules: paracrine factors and adhesion receptors. Stimuli that promote a malignant phenotype can promote additional outcomes in tumor cells, including autophagy and cell death. We summarize here the available evidence about the variables that induce tumor cells to take one or the other of these roads in response to the same stimuli. At the end of this review, we address some unanswered questions in this domain and indicate future directions of research.

Peptide-Mediated Targeting of Cytokines to Tumor Vasculature: The NGR-hTNF Example

A growing body of evidence suggests that the efficacy of cytokines in cancer therapy can be increased by targeting strategies based on conjugation with ligands that recognize receptors expressed by tumor cells or elements of the tumor microenvironment, including the tumor vasculature. The targeting approach is generally conceived to permit administration of low, yet pharmacologically active, doses of drugs, thereby avoiding toxic reactions. However, it is becoming clear that, in the case of cytokines, this strategy has another inherent advantage, i.e. the possibility of administering extremely low doses that do not activate systemic counter-regulatory mechanisms, which may limit their potential therapeutic effects. This review is focused on the use of tumor vasculature-homing peptides as vehicles for targeted delivery of cytokines to tumor blood vessel. In particular, we provide an overview of peptide-cytokine conjugates made with peptides containing the NGR, RGD, isoDGR or RGR sequences and describe, in more details, the biological and pharmacological properties of NGR-hTNF, a peptide-tumor necrosis factor-α conjugate that is currently being tested in phase II and III clinical studies. The results of preclinical and clinical studies performed with these products suggest that peptide-mediated vascular-targeting is indeed a viable strategy for delivering bioactive amounts of cytokines to tumor endothelial cells without causing the activation of counter-regulatory mechanisms and toxic reactions.

Approaches to improve tumor accumulation and interactions between monoclonal antibodies and immune cells

Monoclonal antibodies (mAb) have become a mainstay in tumor therapy. Clinical responses to mAb therapy, however, are far from optimal, with many patients presenting native or acquired resistance or suboptimal responses to a mAb therapy. MAbs exert antitumor activity through different mechanisms of action and we propose here a classification of these mechanisms. In many cases mAbs need to interact with immune cells to exert antitumor activity. We summarize evidence showing that interactions between mAbs and immune cells may be inadequate for optimal antitumor activity. This may be due to insufficient tumor accumulation of mAbs or immune cells, or to low-affinity interactions between these components. The possibilities to improve tumor accumulation of mAbs and immune cells, and to improve the affinity of the interactions between these components are reviewed. We also discuss future directions of research that might further improve the therapeutic efficacy of antitumor mAbs.

Improving drug penetration to curb tumor drug resistance

Several classes of drugs that we refer to here as promoter drugs can improve tumor uptake and penetration of other drugs that we refer to as effector drugs, which exert direct antitumor effects. In this review we discuss the main therapeutic advantages that can be obtained by using promoter drugs. First, tumor-specific enhancement of effector drug accumulation but unaltered accumulation in normal tissues with improvement of the therapeutic index. Second, we propose that curbing tumor drug resistance is another important consequence of using promoter drugs. In particular, we discuss evidence suggesting that promoter drugs can (i) prevent induction of new resistance by paracrine factors released in response to effector drugs, and (ii) reverse existing drug resistance induced by mechanical cues and tumor-cell-extracellular-matrix interactions.

The N-Terminal Fragment of Chromogranin A, Vasostatin-1 Protects Mice From Acute or Chronic Colitis Upon Oral Administration

BackgroundVasostatin-1 (VS-1), the N-terminal fragment of chromogranin A (CgA), decreases the permeability of endothelial cells in vitro and in vivo.AimsHere, we investigated whether a similar effect could be observed also on intestinal epithelial cells (IECs) in vitro and whether VS-1 could have favorable effects on animal models of acute or chronic colitis, which are characterized by increased permeability of the intestinal epithelium.MethodsIn vitro, VS-1 was tested on IEC monolayers showing increased permeability, on mechanically injured IEC monolayers, and on the production of the chemokine IL-8/KC by lipopolysaccharide (LPS)-stimulated IECs. In vivo, VS-1 was tested in animal models of dextran sodium salt (DSS)-induced acute or chronic colitis.ResultsIn vitro, VS-1 inhibited increased permeability of IECs induced by interferon-γ and tumor necrosis factor-α. Moreover, VS-1 promoted healing of mechanically injured IEC monolayers, most likely through stimulation of cell migration, rather than cell proliferation. Eventually, VS-1 inhibited LPS-induced production of IL-8. In vivo, VS-1 exerted protective effects in animal models of acute or chronic colitis upon oral, but not systemic administration.ConclusionsVS-1 is therapeutically active in animal models of acute or chronic, DSS-induced colitis. The mechanisms underlying this effect are likely to be multiple, and may include inhibition of enhanced intestinal permeability, repair of injured intestinal mucosae, and inhibition of the production of IL-8/KC and possibly other inflammatory cytokines.

Intestinal Glucose Uptake Protects Liver from Lipopolysaccharide and d-Galactosamine, Acetaminophen, and Alpha-Amanitin in Mice

We have recently observed that oral administration of D-glucose saves animals from lipopolysaccharide (LPS)-induced death. This effect is the likely consequence of glucose-induced activation of the sodium-dependent glucose transporter-1. In this study, we investigated possible hepatoprotective effects of glucose-induced, sodium-dependent, glucose transporter-1 activation. We show that oral administration of D-glucose, but not of either D-fructose or sucrose, prevents LPS-induced liver injury, as well as liver injury and death induced by an overdose of acetaminophen. In both of these models, physiological liver morphology is maintained and organ protection is confirmed by unchanged levels of the circulating markers of hepatotoxicity, such as alanine transaminase or lactate dehydrogenase. In addition, D-glucose was found to protect the liver from alpha-amanitin-induced liver injury. In this case, in contrast to the previously described models, a second signal had to be present in addition to glucose to achieve protective efficacy. Toll-like receptor 4 stimulation that was induced by low doses of LPS was identified as such a second signal. Eventually, the protective effect of orally administered glucose on liver injury induced by LPS, overdose of acetaminophen, or alpha-amanitin was shown to be mediated by the anti-inflammatory cytokine interleukin-10. These findings, showing glucose-induced protective effects in several animal models of liver injury, might be relevant in view of possible therapeutic interventions against different forms of acute hepatic injury.

Improving Drug Uptake and Penetration into Tumors: Current and Forthcoming Opportunities

The main scope of this topic is to give an update on approaches being studied and developed to improve tumor drug delivery through active targeting and other methods. Inadequate drug accumulation has emerged as one of the main problems underlying therapeutic failure and drug resistance in the treatment of solid tumors (Tredan et al., 2007; Marcucci and Corti, 2012a). It is causally related to the abnormal tumor architecture. Poor vascularization, increased resistance to blood flow and impaired blood supply represent a first obstacle to the delivery of antitumor drugs to tumor cells. Decreased or even inverted transvascular pressure gradients compromise convective transport of drugs. Eventually, an abnormal extracellular matrix offers increased frictional resistance to tumor drug penetration. The net result is reduced overall drug accumulation in tumors, and the propensity of drugs to accumulate in perivascular spaces without penetrating vessel-distant tumor areas. This promotes passive and active induction of drug resistance (Marcucci and Corti, 2012b). n nAbnormal tumor architecture, inadequate drug accumulation and tumor drug resistance are tightly linked phenomena, suggesting that normalization of the tumor architecture, including tumor blood vessels, may result in increased drug delivery to tumors and improve the therapeutic efficacy of anticancer drugs. Indeed, several classes of drugs, that we have referred to as promoter drugs, (Marcucci and Corti, 2012a) have been reported to increase tumor uptake and penetration of antitumor drugs, including drugs that are: (1) vasoactive (Nagamitsu et al., 2009), (2) normalize tumor vessels (Jain, 2005), (3) modify the barrier function of tumor vessels (Corti and Marcucci, 1998; Curnis et al., 2000, 2002), (4) debulk tumor cells (Padera et al., 2004; Moschetta et al., 2012), (5) overcome intercellular (Beyer et al., 2011, 2012; Wang et al., 2011) and stromal barriers (Provenzano et al., 2012). In addition, non-pharmacologic approaches have been described that enhance tumor accumulation of effector drugs (e.g., convection-enhanced delivery, hyperthermia, ultrasound, etc.) (Sen et al., 2011; Watson et al., 2012). n nSome drugs that have already received regulatory approval (e.g., the anti-vascular endothelial growth factor antibody bevacizumab) (Hurwitz et al., 2004) exert antitumor effects at least in part by normalizing the tumor vasculature and enhancing tumor accumulation of chemotherapeutic drugs (Willett et al., 2004). Bevacizumab, however, has a problematic side-effect profile, and the effective doses of the drug encompass a very narrow range beyond which it may even lead to a reduction in drug delivery (Van der Veldt et al., 2012). Additional drugs, acting through other mechanisms of action, are now in clinical development (e.g., vascular targeted NGR-tumor necrosis factor, in phase II/III studies) (Sacchi et al., 2006) and others are about to enter clinical investigation (e.g., Junction Opener-1) (Beyer et al., 2011, 2012). n nTo date, the focus has been primarily on the identification of novel promoter drugs that improve tumor drug delivery. This has led to a considerable number of promoter drugs and devices that are effective in preclinical studies, and some of which have proceeded into clinical investigation or are about to do so. Regarding the types of drugs to be delivered, chemotherapeutics have been the obvious first choice, because they are the antitumor drugs in most widespread use (Curnis et al., 2002; Beyer et al., 2012). Another area of interest is antitumor monoclonal antibodies or related compounds (e.g., immunocytokines) (Beyer et al., 2011; Moschetta et al., 2012), which have become an important component of the antitumor drug armamentarium over the last 15 years. Preclinical investigations have produced promising results when these therapeutic agents are combined with drugs that enhance their penetration into tumors, and it is reasonable to predict that clinical studies will follow in the forthcoming years. So far, so good, but what next? Have we looked at all possible applications for promoter drugs, or are there further applications that we can envisage? We believe that there is still an important field of application for promoter drugs that has been relatively unexplored so far, i.e., the possibility to improve delivery of anticancer cells, in particular immune cells to the tumor (Marcucci et al., 2013), an area of increasing clinical interest. n nEnhancing penetration of immune cells into tumors may have two main therapeutic applications. The first is to improve the efficacy of immune-regulatory antibodies, such as the anti-cytotoxic T-lymphocyte antigen-4 antibody ipilimumab, and the anti-programed death-1 antibody nivolumab. These antibodies yield impressive, and often long-lasting therapeutic responses in a limited fraction (10–20% depending on the antibody) of heavily pretreated patients with metastatic melanoma and other solid tumors (Hodi et al., 2010; Topalian et al., 2012). There is a relationship between the number of tumor-infiltrating immune cells and responsiveness to ipilimumab (Lynch et al., 2012). In this setting, promoter drugs could be of value at two levels: first, to improve tumor delivery of the antibody itself, and second, improve penetration of immune cells into the tumor. This has the potential to increase the fraction of patients that become responders to these antibodies. A second possible field of application are antitumor vaccines. Antitumor vaccines are often active only when administered in a prophylactic setting. With growing tumors, vaccination becomes progressively less effective. One reason might be that tumor-specific lymphocytes become sensitized in draining lymph nodes but are then unable to enter tumors and eliminate tumor cells (Ganss and Hanahan, 1998). Promoter drugs that improve infiltration of immune cells into tumors may prove useful in increasing the effectiveness of cancer vaccines. However, infiltration of immune cells into tumors has requirements that go beyond those of antitumor drugs. Physiological pathways of immune cell extravasation depend on a multistep cascade of events involving tethering, rolling, firm adhesion, and migration. These steps are mediated by distinct adhesion molecules and activation pathways (Springer, 1994); however, adhesion molecules are often downregulated on tumor endothelial cells, a phenomenon defined as endothelial cell anergy (Piali et al., 1995). This impairs the entry of immune cells into tumor sites. In order to enhance tumor infiltration of immune cells, promoter drugs may be required that induce a local inflammatory reaction. This leads to up-regulation of adhesion receptors that are able to attach immune cells to vessel walls and enable their penetration into tumors. Preliminary studies suggest that certain promoter drugs may achieve this goal (Calcinotto et al., 2012). n nPromoter drugs that improve tumor delivery of chemotherapeutics and antitumor antibodies are likely to become a clinical reality in forthcoming years. In addition, new possibilities are emerging to enhance the entrance of therapeutic agents into tumors. For example, recent results suggest that promoter drugs may be useful also for improving infiltration of immune cells into tumors. This may increase the antitumor effects of a broad range of immune-based therapeutics, including immune-regulatory antibodies, antibodies that engage cytotoxic immune cells, and cancer vaccines.