Cellular Uptake and Intracellular Phosphorylation of GS-441524: Implications for Its Effectiveness against COVID-19

GS-441524 is an adenosine analog and the parent nucleoside of the prodrug remdesivir, which has received emergency approval for treatment of COVID-19. Recently, GS-441524 has been proposed to be effective in the treatment of COVID-19, perhaps even being superior to remdesivir for treatment of this disease. Evaluation of the clinical effectiveness of GS-441524 requires understanding of its uptake and intracellular conversion to GS-441524 triphosphate, the active antiviral substance. We here discuss the potential impact of these pharmacokinetic steps of GS-441524 on the formation of its active antiviral substance and effectiveness for treatment of COVID-19. Available protein expression data suggest that several adenosine transporters are expressed at only low levels in the epithelial cells lining the alveoli in the lungs, i.e., the alveolar cells or pneumocytes from healthy lungs. This may limit uptake of GS-441524. Importantly, cellular uptake of GS-441524 may be reduced during hypoxia and inflammation due to decreased expression of adenosine transporters. Similarly, hypoxia and inflammation may lead to reduced expression of adenosine kinase, which is believed to convert GS-441524 to GS-441524 monophosphate, the perceived rate-limiting step in the intracellular formation of GS-441524 triphosphate. Moreover, increases in extracellular and intracellular levels of adenosine, which may occur during critical illnesses, has the potential to competitively decrease cellular uptake and phosphorylation of GS-441524. Taken together, tissue hypoxia and severe inflammation in COVID-19 may lead to reduced uptake and phosphorylation of GS-441524 with lowered therapeutic effectiveness as a potential outcome. Hypoxia may be particularly critical to the ability of GS-441524 to eliminate SARS-CoV-2 from tissues with low basal expression of adenosine transporters, such as alveolar cells. This knowledge may also be relevant to treatments with other antiviral adenosine analogs and anticancer adenosine analogs as well.     

Adenosine triphosphate and adenosine: perspectives in the acute management of paroxysmal supraventricular tachycardia

Adenosine triphosphate (ATP) and adenosine exert strong and transient depressant effects on the sinoatrial and atrioventricular (AV) nodes of the human heart. The AV nodal effects of these drugs explain their high efficacy in either terminating AV re-entrant supraventricular tachycardia or in slowing ventricular rate during atrial tachyarrhythmias. Their very short half-life enables repeated administration of increased doses without reaching toxic effects and explains the transient character of their frequent but benign side effects. These agents represent a good alternative to verapamil in the acute management of paroxysmal supraventricular tachycardia both in infants and adults.     

Adenosine-induced increase in myocardial adenine nucleotides without adenosine-induced systemic hypotension

Summary In anaesthetized rabbits, the i.v. application of 1% adenosine (Ado) for 3 hours at a rate of 4 ml·h^–1·kg^–1 body weight increased the myocardial tissue levels of adenine nucleotides (AN) above the normal values by 39%. This increase in ATP and the sum of AN is a metabolic effect of the continuous and high supply of Ado and does not result from the Ado-induced systemic hypotension: Neither a comparable hypotension and reduction of circulatory work induced by phentolamine nor a massive volume loading caused changes in the AN. The compensation of the Ado-induced hypotension by a simultaneous i.v. application of caffeine or xylometazoline did not interfere with the accumulation of AN. The increase in AN was less pronounced, if norepinephrine was infused to maintain normotension. The increase in AN occurred in left and right ventricular myocardium to a similar extent, although the pressure-volume-work of the left ventricle decreased, and that of the right ventricle increased during Ado-application.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 68 Kardiovaskuläre Restitution und Organsubstitution     

Elevated erythrocyte adenosine deaminase activity in a patient with primary acquired sideroblastic anemia

We report a case of primary acquired sideroblastic anemia (PASA) associated with elevated erythrocyte adenosine deaminase (ADA) activity. The patient was an 85-year-old Japanese male. Analysis of the peripheral blood revealed pancytopenia, and the bone marrow findings showed marked ringed sideroblasts and chromosomal deletion (46XY, 11q-). The erythrocyte ADA activity was 17 times higher than that of normal control, the leukocyte ADA activity was within the normal range, and the plasma ADA activity was 2 times higher than the normal mean. The adenine nucleotides in the patient's erythrocytes were within normal range. According to starch gel electrophoresis, ADA isozyme of the patient was ADA 1. Western blotting showed an increased amount of ADA protein in the patient's erythrocytes. Southern blotting revealed no gene amplification or large structural change. Dot blot analysis of the reticulocyte mRNA showed no increase in the amount of ADA mRNA in the patient's reticulocytes compared with those of reticulocyte-rich controls. We considered that the mechanism of elevated ADA activity in this acquired defect was similar to that found in hereditary hemolytic anemia associated with ADA overproduction.     

腺苷预适应对心脏的保护效应及机制探讨

目的探讨腺苷预适应对心肌缺血/再灌注(I/R)损伤的保护作用.方法通过建立家兔心脏I/R模型,观察腺苷预适应对I/R心肌损伤的保护效应,以左心室功能恢复率、心肌梗死面积、心肌细胞肌酸激酶(CK)和乳酸脱氢酶(LDH)漏出率的变化作为观察指标.结果腺苷预适应组心功能恢复明显好于对照组(P均＜0.01),CK、LDH漏出率及心肌梗死面积均低于单纯I/R组(P均＜0.01),这些心脏保护效应均被腺苷A1受体拮抗剂DPCPX阻断.结论腺苷通过其A1受体的激活介导了缺血预适应对I/R损伤的保护效应.     

On the Mechanism of Action of Theophylline and Caffeine

ABSTRACT Important developments in our understanding of the mechanism of action of methylxan-thines have taken place in the last 10 years. A brief overview of these developments is provided below and the author concludes that the common view that theophylline (and caffeine) acts by raising the levels of cyclic AMP is generally untenable. Instead, many of the actions of the methylxanthines can be explained on the basis of their being antagonists of endogenous adenosine. However the mechanism behind the antiasthmatic effects of xanthines still remains unknown and further research is necessary.     

Prevalence of unidirectional Na+–dependent adenosine transport and altered potential for adenosine generation in diabetic cardiac myocytes

Adenosine is an important physiological regulator of the cardiovascular system. The goal of our study was to assess the expression level of nucleoside transporters (NT) in diabetic rat cardiomyocytes and to examine the activities of adenosine metabolizing enzymes. Isolated rat cardiomyocytes displayed the presence of detectable amounts of mRNA for ENT1, ENT2, CNT1, and CNT2. Overall adenosine (10 μM) transport in cardiomyocytes isolated from normal rat was 36 pmol/mg/min. The expression level of equilibrative transporters (ENT1, ENT2) decreased and of concentrative transporters (CNT1, CNT2) increased in myocytes isolated from diabetic rat. Consequently, overall adenosine transport decreased by 30%, whereas Na⁺–dependent adenosine uptake increased 2–fold, and equilibrative transport decreased by 60%. The activity ratio of AMP deaminase/5'–nucleotidase in cytosol of normal cardiomyocytes was 11 and increased to 15 in diabetic cells. The activity of ecto–5'–nucleotidase increased 2–fold in diabetic cells resulting in a rise of the activity ratio of ecto–5'–nucleotidase/adenosine deaminase from 28 to 56. These results indicate that in rat cardiomyocytes diabetes alters activities of adenosine metabolizing enzymes in such a way that conversion of AMP to IMP is favored in the cytosolic compartment, whereas the capability to produce adenosine extracellularly is increased. This is accompanied by an increased unidirectional Na⁺–dependent uptake of adenosine and significantly reduced bidirectional adenosine transport.     

Enhancement of poly-adenosine diphosphate-ribosylation in human hepatocellular carcinoma

BACKGROUND: Poly-adenosine diphosphate (ADP)-ribosylation, catalysed by poly(ADP-ribose) polymerase (PARP), is a post-translational modification of nuclear proteins and is involved in a wide range of biological processes including DNA repair, cell proliferation and malignant transformation. Alteration of this reaction in human hepatocellular carcinoma (HCC) is of interest, but has not yet been explored. The aim of this study was to evaluate poly-ADP-ribosylation and to compare the expression of PARP in HCC and adjacent non-tumour tissues. METHODS: Tumorous and adjacent non-tumorous tissues were obtained from five consecutive patients with HCC during surgery for tumour resection. Tissue homogenates were subjected to ADP-ribosylation with [32P]-nicotinamide adenine dinucleotide. The ADP-ribosylated proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by autoradiography. Expression of PARP was also evaluated by western blotting. RESULTS: Several proteins were ADP-ribosylated in human HCC tissues. Notably, the radiolabelling of a 116-kDa protein was remarkably greater than that in adjacent non-tumorous tissues (86.5 +/- 35.2 arbitrary units by densitometry vs 12.2 +/- 9.9, mean +/- SD, n = 5, P < 0.02). The radiolabelling of the 116-kDa protein was decreased in the presence of PARP inhibitors in a concentration-dependent manner. Immunoblot analyses revealed that the radiolabelled protein was PARP and that its expression was significantly greater in HCC than in adjacent non-tumorous tissues (333 +/- 204% of non-tumorous tissue, P < 0.05). CONCLUSIONS: We found that poly-ADP-ribosylation and PARP expression were significantly increased in human HCC compared with those in adjacent non-tumorous tissues in surgically obtained specimens.     

Adenosine: trigger and mediator of cardioprotection

Adenosine, a purine nucleoside, is ubiquitous in the body, and is a critical component of ATP. Its concentration jumps 100-fold during periods of oxygen depletion and ischemia. There are four adenosine receptors: A₁ and A₃ coupled to G_i/o and the high-affinity A_2A and low-affinity A_2B coupled to G_s. Adenosine is one of three autacoids released by ischemic tissue which are important triggers of ischemic preconditioning (IPC). It is the A₁ and to some extent A₃ receptors which participate in the intracellular signaling that triggers cardioprotection. Unlike bradykinin and opioids, the other two autacoids, adenosine is not dependent on opening of mitochondrial K_ATP channels or release of reactive oxygen species (ROS), but rather activates phospholipase C and/or protein kinase C (PKC) directly. Another signaling cascade at reperfusion involves activated PKC which initiates binding to and activation of an A₂ adenosine receptor that we believe is the A_2B. Although the latter is the low-affinity receptor, its interaction with PKC increases its affinity and makes it responsive to the accumulated tissue adenosine. A_2B agonists, but not adenosine or A₁ agonists, infused at reperfusion can initiate this second signaling cascade and mimic preconditioning’s protection. The same A_2B receptors are critical for postconditioning’s protection. Thus adenosine is both an important trigger and a mediator of cardioprotection. Returned for 1. revision: 17 September 2007 1. Revision received: 4 October 2007 Returned for 2. revision: 11 October 2007 2. Revision received: 16 October 2007     

Purinergic receptors have no major role in control of the circadian rhythm in rate of thymidine incorporation by cultured chick pineal glands

We have examined the effects of some analogues of adenosine upon the circadian rhythm in rate of thymidine incorporation by cultured chick pineal glands. Incorporation in the early period of the photoperiod on day 2 of culture was slightly inhibited by the adenosine analogue N-ethylcarboxamido-adenosine, but this effect was not countered by the antagonist 8-phenyl-theophylline. Thymidine incorporation was inhibited when glands were continuously exposed to the adenosine transport inhibitor nitrobenzyl-thioinosine, but ongoing incorporation was not inhibited by addition of this agent. Removal of adenosine and deoxyadenosine supplements from the medium, with or without further addition of adenosine deaminase, had no appreciable effects upon thymidine incorporation. We conclude that adenosine and analogues probably play no role in regulation of the rhythm in rate of thymidine incorporation.     

High concentration of adenosine in human visceral leishmaniasis despite increased ADA and decreased CD73

Absence of an effective Th-1 response has been demonstrated as a major cause for the disease pathology among patients with visceral leishmaniasis (VL). Defining strategies to prevent the development of Th-2 response and/or initiate/activate effective Th-1 response may be of help to reduce the growing incidence of drug unresponsiveness. Adenosine, which is considered as an endogenous anti-inflammatory agent is generated in injured/inflamed tissues by the enzymatic catabolism of adenosine triphosphate (ATP), and it suppresses inflammatory responses of essentially all immune cells. The extracellular adenosine-producing pathway comprises two major enzymes CD39 (ATP → ADP → AMP) and CD73 (AMP → Adenosine). In contrast, the adenosine-degrading pathway contains only one major enzyme adenosine deaminase (ADA). Our study shows high concentration of adenosine in diseased condition, varying expression of enzyme involved in adenosine-producing (CD73↓) and adenosine-degrading (ADA↑) pathways. These are less studied in infections like VL but are very important in terms of endogenous regulation of immune response among patients.     

自身免疫性肝病与肝移植

终末期自身免疫性肝病是肝移植的最佳适应证,应综合疾病的特点准确把握移植时机。肝移植术后原发病复发率较高。受排异、胆道并发症等因素的干扰,移植后疾病复发的鉴别诊断难度高,临床医师应重视对疾病复发的认识,及早发现,及时处理。