Current Pharmaceutical Design - Volume 12, Issue 5, 2006

Recently several issues of Current Pharmaceutical Design have been dedicated to cytokines and their clinical applications. The topics on conventional and novel aspects in the field have been extensively covered by the reviews in them. As such, my purpose is to bring up-to-date information on somewhat "atypical" cytokine-related therapies. In this issue of Current Pharmaceutical Design, I invited six experts and their associates to present comprehensive reviews on these emerging topics. I would like to briefly introduce these exciting reviews. Feller and Lewitzky summarize the biology of adapter proteins, with emphasis on Crk and grb2. Both Crk and grb2 are classical adapters of protein-to-protein interaction without known catalytic domains. Alongwith other adapters, CrkL (Crk-like) and Nck, they are involved in signaling, triggered by ligasion of numerous cytokine/growth factor receptors. With 384 references, the readers will find this review exceptionally useful, to keep up with the rapid progresses in our understanding of these adapters and their ligands. Moreover, they also present the potential development of pharmacological reagents, which may modify the protein-to-protein interactions. It is seemingly a challenge, given that these adapters are keys players in so many different signaling pathways. On the other hand, once developed, such reagents may have very widespread applications in diverse clinical settings. Next, Ariga summarizes the development of gene therapy for treatment of primary immunodeficiency [2]. Recombinant cytokines are quite expensive and have only short half life time. Obviously, a gene therapy may be a choice for continual administration of cytokines. Among numerous trials of the gene therapies, those for treatment of immunodeficiency have been most successful. However, as he presents, there appears to be serious inherent problems in the therapy. Recombinant G-CSF has been extensively used to facilitate recovery of peripheral neutrophil counts following chemotherapy and/or bone marrow transplantation. It is also used for mobilization of multipotent immature hematopoietic cells for transplantation. Somewhat atypical, but the most promising utilization of the cytokine is for the treatment of ischemic heart diseases. This review by Komuro's group [3] is a sequel to a review, recently published in Current Pharmaceutical Design [4]. The readers may notice numerous and sometimes-conflicting evidences presented in the fields. Their well-balanced review gives pretty nice pictures of somewhat chaotic situations. Protection of cells from apoptosis is mediated by cytokines. Surprisingly, cilostazol, a well-established, anti-platelet reagent [5], may have a "cytokine-like" function and rescue neuronal cells in ischemic regions. This interesting application of cilostazol is reviewed by Hong's group, who has actually published most of works on this unique aspect of the anti-platelet reagent [6]. Finally, I have also asked two expert groups to summarize two naturally occurring cytokine-like substances. Yatomi, who has established that platelets are a major source of circulating sphingosine 1-phophate, concisely summarizing the diverse functions of the bioactive-lipid [7]. Agonists and antagonists of its receptors are being developed and potential application of these in clinical setting is also described in depth. Kuroki's group calls attention to novel and evolving fields of lung surfactant proteins [8]. The cytokine-like function of these proteins, which regulates the innate immunity, is well presented. They are not just surfactants, but actually are key players in inflammation and immunity in lung tissues. I hope that the readers will really enjoy these reviews on the emerging field of cytokine and anti-cytokine therapies, just as I do. References [1] Feller SM, Lewitzky M. Potential disease targets for drugs that disrupt protein-protein interactions of Grb2 and Crk family adaptors. Curr Pharm Design 2006; 12(5): 529-548. [2] Ariga T. Gene therapy for primary immunodeficiency diseases; recent progress and misgivings. Curr Pharm Design 2006; 12(5): 549-556. [3] Tateno K, Minamino T, Miyauchi H, Kunieda T, Komuro I. Application of Hematopoietis Cells to Therapeutic......

This review summarises some of the knowledge we have about Crk and Grb2 family adaptor protein signalling in health and disease and outlines the current status and the challenges still remaining in the development of efficient and selective inhibitors of their protein - protein interactions. It also highlights briefly some recent successes and problems of inhibitors for proteins that functionally interact with Crk and Grb2 family adaptors, as well as opportunities, which may arise from combination therapies. Grb2 and Crk family adaptors regulate signalling pathways linked to human diseases. They are mainly composed of Src homology 2 (SH2) and Src homology 3 (SH3) domains, which serve as docking sites for signalling proteins, including various receptors, cytoplasmic kinases and GTPase regulators. Considerable insight into the biological functions and mechanisms of action of small SH2/SH3 domain adaptors has been gained in the last years from experimental approaches as diverse as targeted gene disruption and structural studies at the atomic level. This has already indicated several strategies to utilise SH2 and SH3 domain interaction inhibitors in human disease therapy. Additional molecular targets for Crk and Grb2 domain interaction blockers are expected to surface as further protein-protein interactions are discovered. Examples include newly found DOCK family proteins (DOCK3, DOCK4, and DOCK5) which are known or suspected effectors of Crk proteins and the interaction of Grb2 with the cell cycle regulator p27Kip1.

The progress of clinical gene therapy trials during the last two decades has been remarkable, and its application has also expanded into various fields of human diseases. Among them, hereditary diseases such as the primary immunodeficiency diseases (PID) were considered suitable candidates for gene therapy because the therapeutic strategy was very simple, therefore, effective gene therapy may be obtained without significant difficulty compared to other more complex diseases such as cancer. Indeed, the first clinical gene therapy trial was safely performed and was in part, effective for adenosine deaminase (ADA) deficiency patients, a type of severe combined immunodeficiency diseases (SCID). However, because of certain unforeseen obstacles, it took approximately 10 years until the first curative effects were obtained for gene therapy in patients with X-linked SCID (X-SCID). Here, I review and discuss the background and historical events leading up to PID gene therapy, the safety issues, which unexpectedly arose after the successful report, and finally I will attempt to predict the future trends in this form of gene therapy.

Despite considerable progress in the field of cardiovascular medicine and surgery, ischemic heart disease is still the leading cause of death in advanced countries. In this context, it is no wonder why therapeutic angiogenesis, a way to ameliorate ischemic tissue from suffering dysfunction by increasing new blood vessels, gains so much attention from both clinicians and patients. In this review, we will briefly go through a decade of history in therapeutic angiogenesis including unraveling of its mechanisms, results obtained from clinical trials, and lessons learned from earlier investigations. We will then focus on an emerging, yet rapidly evolving field of hematopoietic cell therapy. Recent excellent studies seem to have brought us to the place where we might save so many patients from burden of ischemia, we should be aware that there are some controversies, and sometimes misunderstandings, regarding how or why this treatment does actually work, and what better way should we explore in order to get the best of its efficacy. With these caveats in mind, we will investigate the works elucidating the mechanisms and clinical efficacies of hematopoietic cell therapy.

Cilostazol was developed as a selective inhibitor of cyclic nucleotide phosphodiesterase 3 (PDE3). The antiplatelet and vasodilator properties of cilostazol have been extensively characterized and considered to contribute to the variety of clinical effects such as intermittent claudication and recurrent stroke. In this review, the novel action mechanism (s) of cilostazol are overviewed with the focus on the action of cilostazol in in vitro and in vivo studies as a maxi-K channel opener targeting anti-apoptotic signaling pathways. Under treatment with cilostazol (10 mg/kg intravenously or 30 mg/kg orally), a significant reduction in cerebral infarct area was evident in rats subjected to ischemia/reperfusion. Increase in cyclic AMP and decrease in TNF-α levels were identified in the ipsilateral cortex under treatment with cilostazol accompanied by decreased Bax formation and cytochrome c release with increased Bcl-2 production in the penumbral area as well as in the in vitro human umbilical endothelial cells. Cilostazol suppressed TNF- α-induced decrease in viability of SK-N-SH (human neuroblastoma) cells and HCN-1A (human cortical neuron) cells in association with decrease in PTEN phosphorylation and increase in Akt/CREB phosphorylation with suppression of DNA fragmentation, all of which were antagonized by iberiotoxin, a maxi-K+ channel blocker. Further, cilostazol prevented TNF-α-induced PTEN phosphorylation and apoptotic cell death via increased CK2 phosphorylation in the SK-N-SH cells. Cilostazol increased K+ current in SK-N-SH cells by opening the maxi-K channels. Thus, it was suggested that the action of cilostazol to promote cell survival was ascribed to the maxi-K channel opening-coupled upregulation of CK2 phosphorylation and downregulation of PTEN phosphorylation with resultant increased phosphorylation of Akt and CREB. These in vitro data were confirmed in the in vivo results of rats subjected to focal transient ischemic damage.

Blood platelets are very unique in that they store sphingosine 1-phosphate (Sph-1-P) abundantly (possibly due to the existence of highly active sphingosine kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. Vascular endothelial cells (ECs) and smooth muscle cells (SMCs) respond dramatically to this platelet-derived bioactive lipid mainly through a family of G protein-coupled Sph-1-P receptors named S1P1, 2, 3, 4, and 5, originally referred to as EDG-1, 5, 3, 6, and 8, respectively. In fact, the importance of Sph-1-P in plateletvascular cell interactions has been revealed in a number of recent reports. Through interaction with ECs, Sph-1-P can mediate physiological wound healing processes such as vascular repair, although this important bioactive lipid can become atherogenic and thrombogenic, and cause or aggravate cardiovascular diseases especially under certain pathological conditions. On the other hand, Sph-1-P induces vasoconstriction through interaction with SMCs. It is likely that regulation of Sph-1-P biological activities is important for the therapeutical purpose to control vascular disorders. Particularly, the development of specific S1P receptor agonists or antagonists seems a reasonable strategy to selectively regulate the bioactivity of Sph-1-P, considering that a great diversity of Sph-1-P actions has been reported and that this diversity depends mainly on the S1P receptor subtype involved. In this review, I will summarize recent findings on possible roles of Sph-1-P in vascular biology and its therapeutical implications.

Pulmonary surfactant, a complex of lipids and proteins, functions to keep alveoli from collapsing at expiration. Surfactant proteins A (SP-A) and D (SP-D) belong to the collectin family and play pivotal roles in the innate immunity of the lung. Pulmonary collectins directly bind with broad specificities to a variety of microorganism and possess antimicrobial effects. These proteins also exhibit both inflammatory and anti-inflammatory functions, which occur through interactions with pattern recognition receptors including Toll-like receptor and CD14, signal inhibitory regulatory protein a and a receptor complex of calreticulin and CD91. The collectins enhance phagocytosis of microbes by macrophages through opsonic and/or non-opsonic activities. The proteins stimulate cell surface expression of phagocytic receptors including scavenger receptor A and mannose receptor. Since the expression of SP-A and SP-D is abundant and restricted within the lung, the proteins are now clinically used as biomarkers for lung diseases. The levels of SP-A and SP-D in bronchoalveolar lavage fluids, amniotic fluids, tracheal aspirates and pleural effusions reflect alterations in alveolar compartments and epithelium, and lung maturity. The determination of SP-A and SP-D in sera is a non-invasive and useful tool for understanding some pathological changes of the lung in the diseases, including pulmonary fibrosis, collagen vascular diseases complicated with interstitial lung disease, pulmonary alveolar proteinosis, acute respiratory distress syndrome and radiation pneumonitis.

Tobacco use is the leading risk factor for lung cancer, yet in addition to smoking habit, diet may also play a role in the disease's appearance. While there are reports to indicate that antioxidant vitamins and carotenoids may decrease the risk of lung cancer, results to date have been somewhat ambiguous. This review aimed to describe the results yielded by different studies, which have addressed antioxidant vitamin intake and lung cancer, and to indicate the mechanisms whereby these nutrients might be exercising their activity. Antioxidant vitamins were observed to have no clear protective effect, though there was some evidence pointing to a protective role for vitamins C and E. Vitamin A, in contrast, evinced no clear effect. Insofar as provitamin A carotenoids were concerned, lutein/zeaxanthin, lycopene and alpha-carotene displayed a certain protective trend, yet beta-carotene exhibited no protective effect whatsoever; and indeed, there was speculation as to whether it might even be pernicious in smokers. Beta-criptoxanthin, on the other hand, showed a more consistent protective effect. The study highlighted the need to conduct further research on smokers and non-smokers alike, and in particular, to investigate the effect, if any, on lung cancer of carotenoids or vitamins when ingested in differing dosages.

The calpains represent a well-conserved family of calcium-dependent cysteine proteases. They consist of several ubiquitous and tissue specific isoforms and exhibit broad substrate specificity influencing many aspects of cell physiology including migration, proliferation and apoptosis. Calpain activity in vivo is tightly regulated by its natural endogenous inhibitor calpastatin. Calpastatin specifically inhibits calpain and not other cysteine proteases by interaction with several sites on the calpain molecule. Inappropriate regulation of the calpain-calpastatin proteolytic system is associated with several important human pathological disorders including muscular dystrophy, cancer, Alzheimer's disease, neurological injury, ischaemia/reperfusion injury, atherosclerosis, diabetes and cataract formation. Recent advances in elucidating the tertiary structures of calpain 2 and its regulatory domain calpain 4, together with identification of new modes of regulating calpain activity provide new opportunities for the design of novel calpain inhibitors. Several classes of inhibitors, including peptidyl epoxide, aldehyde, and ketoamide inhibitors, targeting the active site have proven effective against the calpains and are in the process of evaluation in animal models of human disease. However, a major limitation to the clinical use of such inhibitors is their lack of specificity among cysteine proteases and other proteolytic enzymes. The development of a new class of calpain inhibitors that interact with domains outside of the catalytic site of calpain may provide greater specificity and therapeutic potential.

Recent glycobiology studies have suggested fundamental biological functions for chondroitin sulfate (CS) and dermatan sulfate (DS), which are widely distributed as glycosaminoglycans (GAGs) sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. Several biological functions are closely associated with the structure and in particular with the sulfation patterns of these polysaccharides. CS is also used as a structure-modifying osteoarthritis (OA) drug that reverses, retards, or stabilizes the pathology of OA, thereby providing symptomatic relief in the long-term treatment. Advances in analytical separational techniques, including agarose-gel electrophoresis, high-performance liquid chromatography (HPLC), capillary electrophoresis (HPCE), fluorophore-assisted carbohydrate electrophoresis (FACE) and electrospray ionization mass (ESI-MS) enable us to examine alterations of CS/DS with respect to their quantities and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. Furthermore, sensitive analytical procedures enable us to follow the pharmacological application of CS in the treatment of OA and to monitor the progression of the disorder. In this review, the chromatographic and electromigration procedures developed to analyse and characterise CS/DS are presented. Moreover, a critical evaluation of the biological relevance of the results obtained by the developed methodology is discussed.