Delamanid: From discovery to its use for pulmonary multidrug-resistant tuberculosis (MDR-TB).

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is the leading cause of death from an infectious disease globally. The widespread and ever-increasing resistance to TB drugs is reducing the effectiveness of treatment and jeopardizing TB control. New effective drugs with acceptable safety profiles are needed to turn the tide. Since the early 1990s, Otsuka Pharmaceutical Co., Ltd. has had a TB drug development program that resulted in the selection and development of delamanid (OPC-67683, Deltyba®), a first-in-class bicyclic nitroimidazole. Delamanid was initially approved by the European Medicines Agency (EMA) in 2014 for the treatment of adult pulmonary multi-drug resistant (MDR)-TB when an effective treatment regimen cannot otherwise be composed for reasons of resistance or tolerability. It has since been approved by several other countries/regions. In this review, we describe the history of delamanid's development, including the screening process, in vitro and in vivo characterization, as well as various clinical studies. Delamanid possesses potent activity against replicating, dormant, and intracellular MTB bacilli, and is bactericidal in mouse and guinea pig TB models. Delamanid resistance mechanisms have been attributed to genes in the F420-dependent deazaflavin nitroreductase bio-activation pathway, found in mycobacterium species but not in common bacterial or mammalian cells. Published susceptibility testing results from 744 clinical isolates from delamanid-naïve patients indicate that the natural resistance rate to delamanid is very low (1.3%). Delamanid is largely metabolized by albumin in serum, and to a much less extent by cytochrome P450 enzymes. Furthermore, it neither inhibits nor induces P450 enzymes. In terms of efficacy, delamanid demonstrated activity in an early bactericidal activity trial in drug susceptible pulmonary TB patients and increased 2-month sputum culture conversion rates when added to an optimized background regimen in MDR-TB patients in a phase 2b global clinical trial. In addition, recent results outside clinical studies show favourable responses in highly resistant TB patients including extensively drug resistant (XDR)-TB when treated with delamanid-containing regimens in routine programmatic settings. The primary safety concern with delamanid is QTcF interval prolongation, although this observation has thus far not been associated with any clinical cardiac events. Overall, delamanid appears to be a well-tolerated and safe anti-TB drug when compared to other drugs used to treat MDR-TB.

Cilostazol increases tissue blood flow in contracting rabbit gastrocnemius muscle.

BACKGROUND: The mechanisms underlying the ability of cilostazol to improve walking distance in patients with intermittent claudication (IC) are not fully understood, but may be related to its phosphodiesterase type 3 (PDE3) and adenosine uptake inhibition. In the present study the effect of cilostazol on blood flow and interstitial adenosine concentration was compared with that of the PDE3 inhibitor, milrinone, and the adenosine uptake inhibitor, draflazine. METHODS AND RESULTS: Rabbit gastrocnemius muscle blood flow was measured under resting, contracting and ischemic conditions. Interstitial adenosine was sampled by microdialysis. None of the drugs affected tissue blood flow at rest. Blood flow in electrically stimulated muscle was 2- to 3-fold higher in vehicle-, milrinone- and draflazine-treated animals. However, cilostazol caused an 8-fold increase. Ligation of the femoral artery decreased blood flow in the stimulated muscle in all groups to a similar degree. Cilostazol and draflazine increased the dialysate adenosine concentration during the first 10 min of muscle contraction, but had no effect during ischemia, most likely because of the high AMP deaminase activity in skeletal muscle. CONCLUSIONS: Cilostazol increases blood flow in the gastrocnemius muscle during contraction and it is this effect that may be partially responsible for the improved walking distance in IC patients. (Circ J 2010; 74: 181 - 187).

Highly potent anti-human GPVI monoclonal antibodies derived from GPVI knockout mouse immunization.

Recent progress in the understanding of thrombus formation has suggested an important role for glycoprotein (GP) VI in this process. To clarify the exact role in detail, it is necessary to use specific, high affinity inhibitory antibodies. However, possibly due to the conserved structure of GPVI among species, it has been difficult to obtain potent antibodies. In this study, we developed highly potent anti-human GPVI monoclonal antibodies using GPVI knockout mice for immunization. Fab fragments of these antibodies, named OM1 and OM2, potently inhibit collagen-induced platelet aggregation. The IC(50) values for OM1 and OM2 are 0.6+/-0.05 and 1.7+/-0.5 microg/mL, respectively, showing potency greater than, or equal to that of abciximab (1.7+/-0.3 microg/mL), an anti-GPIIb/IIIa antibody. Fab fragments of OM1 and OM2 also potently inhibit collagen-induced ATP release, thromboxane A(2) formation, and platelet adhesion to immobilized collagen under static and flow conditions. Interestingly, platelet aggregation induced with collagen-related peptide was potently inhibited by OM2 but not OM1, indicating that OM1 recognizes an epitope that is different from collagen-related peptide-binding site on GPVI. These results suggest that OM1 and OM2 may be useful tools to understand the role of GPVI in thrombus formation. Furthermore, these antibodies have the potential to be developed as a new class of therapeutic tool.

GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism.

Platelet glycoprotein VI (GPVI) is now considered to be a major player in platelet-collagen adhesive interactions leading to thrombus formation. GPVI blockade, or its depletion, has been shown in mice to result in complete protection against arterial thrombosis, without significant prolongation of bleeding time. GPVI may therefore represent a useful antithrombotic target. In order to reaffirm the role of GPVI in platelet-collagen interactions, we developed GPVI(null) mice by targeted disruption methodology. GPVI(null) mice platelets failed to respond to a high dose of fibrillar collagen, or convulxin, a GPVI agonist, but showed a normal response to other agonists such as ADP, PMA and arachidonic acid. We report, for the first time, that a proportion of GPVI(null) mice is protected against lethal thromboembolism, induced by the infusion of a mixture of collagen and epinephrine. Greater than 55% of GPVI(null) mice survived the challenge, whereas the maximal survival from the other genotypes was 17% (n=18 per genotype). Washed platelets obtained from GPVI(null) mice showed >90% reduction in adhesion to fibrillar collagen under static conditions. Platelet adhesion to collagen under dynamic conditions using a high shear rate (2600 s(-1)) was dramatically reduced using blood from GPVI(null) mice, while platelets from wild-type and heterozygous animals showed a similar amount of adhesion. Animals from each genotype had essentially similar tail bleeding time, suggesting that a complete deficiency of GPVI, at least in mice, does not result in an enhanced bleeding tendency. These observations clearly establish that blockade of GPVI may attenuate platelet-collagen interactions without adversely affecting the bleeding time.

Antiplatelet and antithrombotic activity of cilostazol is potentiated by dipyridamole in rabbits and dissociated from bleeding time prolongation.

PURPOSE: To determine the antiplatelet effect of cilostazol (Pletal) and its interaction with dipyridamole in in vitro and in vivo rabbit models, and to see if it can be dissociated from bleeding time prolongation. METHODS: In vitro collagen-induced platelet aggregation was measured by an impedance-based aggregometer. The in vivo antithrombotic effect was evaluated in a rabbit carotid artery cyclic flow reduction (CFR) model, in which repetitive thrombosis was induced by mechanical injuries of the artery and stenosis. Template bleeding time was determined in rabbit ear arterioles and hindlimb nail cuticles. RESULTS: In vitro platelet aggregation was slightly inhibited by 4 microM cilostazol (22 +/- 6%), and modestly by 13 microM (57 +/- 3% of aggregation). While dipyridamole itself up to 13 microM had no significant inhibition, it potentiated the effect from cilostazol: in the presence of 4 microM dipyridamole, 4 microM cilostazol inhibited aggregation by 47 +/- 6%. Dipyridamole also potentiated the CFR reducing effect of cilostazol: combination of dipyridamole (no effect by itself) and cilostazol at 1 microM decreased CFRs to levels achieved by 3-4 microM cilostazol alone. Bleeding times were similar in controls and animals treated with cilostazol, or with cilostazol and dipyridamole. In contrast, aspirin (4 mg/kg), while reducing CFRs, significantly increased bleeding time. CONCLUSION: These results suggest that dipyridamole potentiates the antiplatelet effect of cilostazol without prolongation of the bleeding time, implying a potential novel combination antithrombotic therapy.

Cilostazol and dipyridamole synergistically inhibit human platelet aggregation.

It has been previously shown that cilostazol (Pletal), a drug for relief of symptoms of intermittent claudication, potently inhibits cyclic nucleotide phosphodiesterase type 3 (PDE3) and moderately inhibits adenosine uptake. It elevates extracellular adenosine concentration, by inhibiting adenosine uptake, and combines with PDE3 inhibition to augment inhibition of platelet aggregation and vasodilation while attenuating positive chronotropic and inotropic effects on the heart. In the present study, we tested the hypothesis that cilostazol combined with a more potent adenosine uptake inhibitor, dipyridamole, synergistically inhibited platelet aggregation in human blood. In the presence of exogenous adenosine (1 microM), the combination of cilostazol and dipyridamole synergistically increased intra-platelet cAMP. Furthermore, cilostazol inhibited platelet aggregation in a washed platelet assay concentration-dependently with IC50s of 0.17 +/- 0.04 microM (P < 0.05 versus plus adenosine alone of 0.38 +/- 0.05 microM), 0.11 +/- 0.06 microM (P < 0.05), and 0.01 +/- 0.01 microM (P < 0.005) when combined with 1, 3, or 10 microM dipyridamole, respectively (n = 5). In whole blood, cilostazol (0.3 to 3 microM) and dipyridamole (1 or 3 microM) synergistically inhibited collagen- and ADP-induced platelet aggregation in vitro. Furthermore, the synergism was confirmed in an open-label, sequential study in healthy human subjects using ex vivo whole-blood collagen-induced platelet aggregation. Four hours after oral co-administration of cilostazol (100 mg) and dipyridamole (200 mg), platelet aggregation was inhibited by 45 +/- 17%, while no significant inhibition was observed from subjects treated with either drug alone. The combination may provide a potential treatment of arterial thrombotic disorders.

Cilostazol as a unique antithrombotic agent.

Cilostazol (CLZ) was originally developed as a selective inhibitor of cyclic nucleotide phosphodiesterase 3 (PDE3). PDE3 inhibition in platelets and vascular smooth muscle cells (VSMC) was expected to provide an antiplatelet effect and vasodilation. Recent preclinical studies have demonstrated that CLZ also possesses the ability to inhibit adenosine uptake by various cells, a property that distinguishes CLZ from other PDE3 inhibitors, such as milrinone. After extensive preclinical and clinical studies, CLZ has been shown to have unique antithrombotic and vasodilatory properties based upon these novel mechanisms of action. CLZ was approved in 1988 for the treatment of symptoms related to peripheral arterial occlusive disease in Japan (Pletaal) and in 1999 in the U.S. and in 2001 in the U.K. (Pletal) for the treatment of intermittent claudication symptoms. Despite its remarkable antiplatelet properties, CLZ is not generally considered an antithrombotic agent in Western countries, perhaps due to the bulk of its antithrombotic preclinical and clinical development being conducted in Japan. In this review, the unique properties of CLZ are reviewed with the focus on CLZ as a unique antiplatelet agent targeting platelets and VSMC, demonstrating synergy with endogenous mediators and showing lowered risk of bleeding risk compared to other antiplatelet drugs.

Expression of glycoprotein VI in vascular endothelial cells.

Glycoprotein (GP) VI, a collagen receptor, plays a important role in collagen-mediated platelet aggregation and adhesion. To date, GPVI expression has been found only in platelets and megakaryocytes. In the present studies, we have demonstrated that GPVI was also expressed in cultured human umbilical vein endothelial cells (HUVEC) at both transcript and protein levels. Using a GPVI-specific probe, a approximately 6-kb band was detected in HUVEC as well as in platelets and megakaryoblastic cell lines by Northern blotting. Using polyclonal antibodies raised against platelet GPVI peptides, the same size band (57 kDa) was labeled with convulxin (CVX) after immuo-precipitation in both HUVEC and platelet lysates. In addition, a approximately 70-kDa band was also labeled in HUVEC. Surface expression of GPVI in HUVEC was confirmed by flow cytometry with GPVI-specific IgG or by direct labeling with FITC-conjugated CVX. Since HUVEC lack FcRgamma chain that forms complex with GPVI in platelets for signaling process, the function of GPVI in vascular endothelial cells remains to be determined.

Expression of glycoprotein VI in vascular endothelial cells.

Glycoprotein (GP) VI, a collagen receptor, plays an important role in collagen-mediated platelet aggregation and adhesion. To date, GPVI expression has been found only in platelets and megakaryocytes. In the present studies, we have demonstrated that GPVI was also expressed in cultured human umbilical vein endothelial cells (HUVEC) at both transcript and protein levels. Using a GPVI-specific probe, a ˜6-kb band was detected in HUVEC as well as in platelets and megakaryoblastic cell lines by Northern blotting. Using polyclonal antibodies raised against platelet GPVI peptides, the same size band (57 kDa) was labeled with convulxin (CVX) after immuno-precipitation in both HUVEC and platelet lysates. In addition, a ˜70-kDa band was also labeled in HUVEC. Surface expression of GPVI in HUVEC was confirmed by flow cytometry with GPVI-specific IgG or by direct labeling with FITC-conjugated CVX. Since HUVEC lack FcRγ chain that forms complex with GPVI in platelets for signaling process, the function of GPVI in vascular endothelial cells remains to be determined.

New mechanism of action for cilostazol: interplay between adenosine and cilostazol in inhibiting platelet activation.

Cilostazol, a potent phosphodiesterase 3 inhibitor and anti-thrombotic agent, was recently shown to inhibit adenosine uptake into cardiac myocytes and vascular cells. In the present studies, cilostazol inhibited [ H]-adenosine uptake in both platelets and erythrocytes with a median inhibitory concentration (IC ) of 7 micro M. Next collagen-induced platelet aggregation was studied and it was found that adenosine (1 micro M ), having no effect by itself, shifted the IC of cilostazol from 2.66 micro M to 0.38 micro M (p < 0.01). This shifting was due to an enhanced accumulation of cAMP in platelets and was significantly larger than that by the combination of adenosine and milrinone, which has no effect on adenosine uptake. Similarly, cilostazol, by blocking adenosine uptake, enhanced the adenosine-mediated cAMP increase in Chinese hamster ovary cells that overexpress human A receptor. Furthermore, the inhibitory effect of cilostazol on platelet aggregation in whole blood was significantly reversed by ZM241385 (100 n ), an A adenosine receptor antagonist, and by adenosine deaminase (2 U/ml). These data suggest that the inhibitory effects of cilostazol on adenosine uptake and phosphodiesterase 3 together elevate intracellular cAMP, resulting in greater inhibition of agonist-induced platelet activation.

Comparison of the effects of cilostazol and milrinone on cAMP-PDE activity, intracellular cAMP and calcium in the heart.

We investigated the basis for the difference in the cardiotonic effects of the PDE3 inhibitors cilostazol and milrinone in the rabbit heart. Cilostazol displayed greater selectivity than milrinone for inhibition of cAMP-PDE activity in microsomal vs cytosolic fractions from rabbit heart. This difference was due to the inhibition of significantly less cytosolic cAMP-PDE activity by cilostazol compared to milrinone. A combination of cilostazol (>15 microM) and the PDE4 selective inhibitor, rolipram (5 microM), inhibited levels of cytosolic cAMP-PDE activity similar to those inhibited by milrinone on its own. This suggested that milrinone inhibited PDE4 in addition to PDE3 activity. In isolated rabbit cardiomyocytes, milrinone (>10 microM) caused greater elevations in intracellular cAMP and calcium than cilostazol. In the presence of rolipram, however, the cAMP and calcium elevating effects of cilostazol and milrinone were similar. Therefore, in rabbit heart, partial inhibition of PDE4 by milrinone contributed to greater increases in cardiomyocyte cAMP and calcium levels than cilostazol. PDE4 activity in failing human heart was lower than in rabbit heart and there was no significant difference in the inhibition of human cytosolic cAMP-PDE by cilostazol and milrinone. Our results suggest that in normal rabbit heart inhibition of PDE4 by milrinone may partly contribute to the greater cardiotonic effect of milrinone when compared to cilostazol. However, the lower level of PDE4 activity in failing human heart suggests that factors other than inhibition of PDE4 by milrinone may contribute to differences in cardiotonic action when compared to cilostazol.