Use of dextran 75 as a macromolecular agent in centrifugal leukapheresis

Twenty-five leukaphereses were done by the Haemonetics system using citrated Dextran 75 in normal saline as anticoagulant, and were compared with a much larger series using hydroxyethyl starch (HES) with and without steroid premedication. The granulocyte and platelet yields were only minimally different between the two agents. Both were inferior in granulocyte collection to the group that was premedicated with a steroid. Dextran is thus a satisfactory substitute for HES in leukapheresis with this system.     

Tea catechins as a potential alternative anti-infectious agent

Besides well-known health benefits, green tea catechins exert antimicrobial and antiviral activities against a variety of infectious agents. Although the detailed mechanism of the antimicrobial activity of tea catechins remains to be explored, the broad-spectrum activity of catechins may involve common target(s), such as the cell membrane, in addition to specific targets for each pathogen. This extends to antiviral activities, where many pronounced activities were reported for enveloped viruses. Yet, the effectiveness of tea catechins as antimicrobials is compromised by relative chemical instability and poor bioavailability. Whether tea catechins will emerge as a viable option as alternative medicine or as a synergistic combination therapy with pre-existing antivirals or antibiotics must therefore depend on a method of delivery that ensures its stability and bioavailability. However, green tea may provide an option for mitigating the health and economic burdens associated with emerging and re-emerging infectious diseases, especially considering the paucity of effective control measures. Considering the zoonotic nature of newly arising infectious diseases, the dual use of green tea components in both humans and livestock may reduce animal–human transmission, which would complement the current management of infectious diseases.     

Tea catechins as a potential alternative anti-infectious agent

Besides well-known health benefits, green tea catechins exert antimicrobial and antiviral activities against a variety of infectious agents. Although the detailed mechanism of the antimicrobial activity of tea catechins remains to be explored, the broad-spectrum activity of catechins may involve common target(s), such as the cell membrane, in addition to specific targets for each pathogen. This extends to antiviral activities, where many pronounced activities were reported for enveloped viruses. Yet, the effectiveness of tea catechins as antimicrobials is compromised by relative chemical instability and poor bioavailability. Whether tea catechins will emerge as a viable option as alternative medicine or as a synergistic combination therapy with pre-existing antivirals or antibiotics must therefore depend on a method of delivery that ensures its stability and bioavailability. However, green tea may provide an option for mitigating the health and economic burdens associated with emerging and re-emerging infectious diseases, especially considering the paucity of effective control measures. Considering the zoonotic nature of newly arising infectious diseases, the dual use of green tea components in both humans and livestock may reduce animal-human transmission, which would complement the current management of infectious diseases.     

Radiolabeled albumin through SNAr of cysteines as a potential pretargeting theranostic agent

Human serum albumin (HSA) has been shown to be a promising tumor targeting vector and target for generating theranostics by bioconjugation. Unstable chemical conjugation to HSA via a cysteine (Cys34) by reversible Michael additions is most commonly applied for this purpose. Herein, we describe utilization of our recently developed site-selective irreversible S_NAr conjugation to Cys34 using perfluorobenzene sulfonyl derivatives to introduce a trans-cyclooctene (TCO) handle. The TCO could then be bioorthogonally ligated within minutes through an inverse-electron demand Diels–Alder reaction (IEDDA) to tetrazines (Tzs) containing a radionuclide. The methodology opens up a wide range of chemistries including pretargeting, ‘click-to-release’ tumor selective drug delivery or ultra-fast and complete conjugation of any drug. The proof-of-principle study demonstrated that the conjugation chemistry is feasible, robust and easy to carry out, being promising for pretargeted imaging and therapy studies as well as selective drug delivery using HSA.

Human serum albumin has been functionalized with a radionuclide by combining S_NAr conjugation to Cys34 with CuAAC and inverse-electron demand Diels–Alder reactions demonstrating a promising strategy for generating theranostics by bioconjugation.     

Hyaluronic acid–green tea catechin conjugates as a potential therapeutic agent for rheumatoid arthritis

Fibroblast-like synoviocytes are a key effector cell type involved in the pathogenesis of rheumatoid arthritis. The major green tea catechin, epigallocatechin-3-O-gallate (EGCG), has attracted significant interest for rheumatoid arthritis therapy because of its ability to suppress the proliferation and interleukin-6 secretion of synoviocytes. However, therapeutic efficacy of EGCG has been limited by a lack of target cell specificity. Herein we report hyaluronic acid–EGCG (HA–EGCG) conjugates as an anti-arthritic agent that is capable of targeting fibroblast-like synoviocytes via HA–CD44 interactions. These conjugates exhibited superior anti-proliferative and anti-inflammatory activities compared with EGCG under simulated physiological conditions. Near-infrared fluorescence imaging revealed preferential accumulation of the conjugates at inflamed joints in a collagen-induced arthritis rat model, and their anti-arthritic efficacy was investigated by measuring a change in the edema and histopathological scores. Our findings suggest the potential of HA–EGCG conjugates as an anti-arthritic agent for the treatment of rheumatoid arthritis.

Macromolecular HA–EGCG conjugates undergo targeted internalization by CD44-overexpressing fibroblast-like synoviocytes and subsequently cause H₂O₂-induced cell death and inhibition of IL-6 secretion, thereby suppressing the progression of arthritis.     

Modified fluid gelatin. An alternative macromolecular agent for centrifugal leukapheresis

We studied a French modified fluid gelatin (MFG), substituting it for hydroxyethyl starch (HES) in leukapheresis procedures using three currently available blood cell separators, and observing its effects on the function of platelets and granulocytes. As a cell-collecting agent, we found MFG to be as effective as HES with intermittent flow centrifugation (Haemonetics), and slightly less so with one continuous flow device (IBM 2997). MFG was clearly less effective than HES with the Fenwal CS-3000 continuous flow separator, although we have reason to believe it would be possible to improve efficiency with this machine by changing the operating variables. Tests of platelet and granulocyte function showed negligible alteration with either agent and no difference between them. MFG disappears much more rapidly from the circulation than HES (after a single injection, it is undetectable by the third day). Reaction frequency with MFG was about the same as that of HES, with perhaps somewhat more frequent allergic manifestations.     

8-amino-adenosine is a potential therapeutic agent for multiple myeloma

Multiple myeloma (MM) is a malignancy of clonal B-cells that accounts for 10% of all hematologic malignancies. We have shown previously that a novel purine analogue, 8-chloro-adenosine, has significant activity for MM in preclinical studies. OBJECTIVE: Using MM cell lines, we investigated the molecular mechanism of related congener of adenosine, 8-amino-adenosine (8-NH2-Ado). METHODS: We employed biological and biochemical assays in MM cell lines to evaluate the clinical potential of 8-NH2-Ado. RESULTS: In MM cell lines both sensitive and resistant to conventional chemotherapies, 8-NH2-Ado is cytotoxic, with IC50 ranging from 300 nmol/L to 3 micromol/L. A mouse leukemic cell line lacking adenosine kinase activity was resistant to 8-NH2-Ado, indicating that phosphorylation of 8-NH2-Ado to its triphosphate form is required for cytotoxicity. A 4-hour incubation of MM cells with 10 micromol/L analogue resulted in an accumulation of >7 mmol/L 8-NH2-ATP with a parallel decline in the endogenous ATP levels. Accumulation of 8-NH2-ATP was dependent on both exogenous concentration of 8-NH2-Ado and incubation time. The accumulation of 8-NH2-ATP was accompanied by a decrease in both RNA and DNA synthesis. The mechanism of 8-NH2-Ado-mediated cytotoxicity was due to apoptosis as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a sub-G1 DNA content. CONCLUSION: Based on these results, we conclude that 8-NH2-Ado may hold great potential as a therapeutic agent for the treatment of MM.     

Fibrinogen concentrate – a potential universal hemostatic agent

Background: Human fibrinogen concentrates have been commercially available for decades for substitution therapy in hypofibrinogenemia, dysfibrinogenemia and afibrinogenemia. Accumulating new data suggest that fibrinogen plays a critical role in achieving and maintaining hemostasis, particularly in patients suffering from acquired fibrinogen deficiency during massive bleeding, where benefit from early intervention with fibrinogen concentrate appears to be important. Objective/methods: This work focuses on pasteurized fibrinogen concentrate, with special emphases on product characteristics, pharmacodynamics, pharmacokinetics, laboratory monitoring, dosing, clinical efficacy, safety and tolerability. Future clinical and laboratory perspectives on fibrinogen are discussed and outlined. Results/conclusion: Pasteurized fibrinogen concentrate is derived from human plasma. Its half life is 2.7 days in patients with congenital fibrinogen deficiency. For congenital and acquired deficiency in vivo recovery rates vary from 60% to 109%. Reportedly, administration of pasteurized fibrinogen in patients with congenital deficiency is efficacious. Acquired deficiency of fibrinogen appears to be an early event in seriously bleeding patients, preceding critical levels of platelets or other coagulation factors. Experimental animal studies, as well as clinical observations suggest a beneficial role of early substitution with fibrinogen in management of critical traumatic and surgical bleeds. Pasteurized fibrinogen concentrate is well tolerated and associated with a low incidence of adverse thrombo-embolic events.     

Enzymic assay for oxalate in unprocessed urine, as adapted for a centrifugal analyzer

In this automated modification of the oxalate decarboxylase method, oxalate can be measured (12 per hour) in acidified but otherwise unprocessed urine. Standard curves are linear up to at least 2.5 mmol/L. When 0.50 mmol of oxalate was added per liter to samples of 18 patients' urines, a mean analytical recovery of 98.5% (SD 3.6%) was obtained. Within-series CVs were 3.4 and 1.0%, between-series CVs 7.3 and 2.7% (n = 15) for oxalate concentrations of 0.31 and 0.61 mmol/L. The lower limit of detection is 25 mumol/L. Concentrations measured with this "direct" method correlated well (r = 0.95) with those measured after precipitation with calcium and ethanol and resolubilization in dilute sulfuric acid. For 17 healthy volunteers the mean urinary excretion of oxalate was 0.37 (SD 0.14) mmol/24 h.     

MK-4815, a potential new oral agent for treatment of malaria

Malaria continues to have a significant impact on the health of the developing world. Efforts to combat this disease now focus on combination therapy in order to stem the emergence of resistant parasites. Continued efforts are needed to discover and develop new agents for use in combination antimalarial regimens. MK-4815 is a small molecule with antimalarial activity that was identified from a large pharmaceutical compound collection using a semiautomated version of a well-established in vitro assay for the erythrocytic stages of Plasmodium falciparum. In vitro studies indicate that the compound selectively accumulates in infected red blood cells and is most effective against the metabolically active late trophozoite/early schizont stages. A variety of drug-resistant field isolates of P. falciparum were found to be as sensitive to MK-4815 as the wild-type lines. MK-4815 is orally active in a P. berghei mouse model of acute malaria. In this model, where untreated animals succumb to infection 10 to 12 days postinfection, MK-4815 was completely curative when given as a single dose of 50 mg/kg, 2 doses of 25 mg/kg, or 4.5 doses of 12.5 mg/kg. In pharmacokinetic studies with mice and rhesus monkeys, MK-4815 demonstrated oral bioavailability and low clearance. In addition, MK-4815 is inexpensive to synthesize, an important characteristic for providing affordable antimalaria therapy to the developing world. The attractive biological and pharmaceutical profile of MK-4815 demonstrates its potential for use in combination with other agents in the fight against malaria.     

Polyphosphate as a general procoagulant agent

Summary. Background: Polyphosphate is secreted by activated platelets and we recently showed that it accelerates blood clotting, chiefly by triggering the contact pathway and promoting factor (F) V activation. Results: We now report that polyphosphate significantly shortened the clotting time of plasmas from patients with hemophilia A and B and that its procoagulant effect was additive to that of recombinant FVIIa. Polyphosphate also significantly shortened the clotting time of normal plasmas containing a variety of anticoagulant drugs, including unfractionated heparin, enoxaparin (a low molecular weight heparin), argatroban (a direct thrombin inhibitor) and rivaroxaban (a direct FXa inhibitor). Thromboelastography revealed that polyphosphate normalized the clotting dynamics of whole blood containing these anticoagulants, as indicated by changes in clot time, clot formation time, alpha angle, and maximum clot firmness. Experiments in which preformed FVa was added to plasma support the notion that polyphosphate antagonizes the anticoagulant effect of these drugs via accelerating FV activation. Polyphosphate also shortened the clotting times of plasmas from warfarin patients. Conclusion: These results suggest that polyphosphate may have utility in reversing anticoagulation and in treating bleeding episodes in patients with hemophilia.     

Adenosine as a therapeutic agent.

Adenosine, an endogenous nucleoside has been recently approved for use in the treatment of paroxysmal supraventricular tachycardia. Adenosine is nearly 100% effective in terminating tachycardia in which the atrioventricular node forms part of the reentrant circuit. Although most ventricular tachycardias are insensitive to adenosine, this substance is effective in ventricular tachycardia induced by catecholamines or exercise. An intravenous bolus dose of 6 mg is the initial dose. If no effect is noted a further bolus of 12 mg can be given. The most common side effects are dyspnea, chest pressure and facial flushing. This article reviews, in addition, some of the comparative trials with verapamil and adenosine triphosphate, some of the additional therapeutic indications, the possible mechanisms of action in cardiac tissue, and the type of purinergic receptors involved in the antiarrhythmic effects of adenosine.     

Circumsporozoite protein as a potential target for antimalarials

Since the discovery of circumsporozoite protein (CSP), a major sporozoite surface antigen, by Ruth Nussenzweig and Victor Nussenzweig in the early 1980s, the role of CSP in protection against malaria has been extensively investigated. Several monoclonal antibodies against CSP have been generated to date, with some of them mediating antimalarial protection upon passive transfer into animals. Genetically engineered transgenic mosquitoes producing the anti-CSP antibody have recently been generated to reduce malarial transmission. A monoclonal anti-CSP antibody was produced in mice by adeno-associated virus vector, which protected them from malaria. Phase III trials with RTS,S vaccine that targets CSP of Plasmodium falciparum have shown modest efficacy. Polyclonal anti-CSP antibodies derived from children who received the RTS,S vaccine failed to block malarial transmission through mosquitoes, but passive transfer of monoclonal antibodies raised from RTS,S-vaccinated recipient conferred protection against malaria in mice. Taken together, these findings may imply CSP as an antimalarial target.