Cystic fibrosis improves COVID-19 survival and provides clues for treatment of SARS-CoV-2.

Systemic pools of ATP are elevated in individuals homozygous for cystic fibrosis (CF) as evidenced by elevated blood and plasma ATP levels. This elevated ATP level seems to provide benefit in the presence of advanced solid tumors (Abraham et al., Nature Medicine 2(5):593-596, 1996). We published in this journal a paper showing that IV ATP can elevate the depleted ATP pools of advanced cancer patients up to levels found in CF patients with subsequent clinical, biochemical, and quality of life (QOL) improvements (Rapaport et al., Purinergic Signalling 11(2): 251-262, 2015). We hypothesize that the elevated ATP levels seen in CF patients may be benefiting CF patients in another way: by improving their survival after contracting COVID-19. We discuss here the reasoning behind this hypothesis and suggest how these findings might be applied clinically in the general population.

Continuous intravenous infusion of ATP in humans yields large expansions of erythrocyte ATP pools but extracellular ATP pools are elevated only at the start followed by rapid declines.

The pharmacokinetics of adenosine 5'-triphosphate (ATP) was investigated in a clinical trial that included 15 patients with advanced malignancies (solid tumors). ATP was administered by continuous intravenous infusions of 8 h once weekly for 8 weeks. Three values of blood ATP levels were determined. These were total blood (erythrocyte) and blood plasma (extracellular) ATP pools along with the initial rate of release of ATP into the blood plasma. We found that values related to erythrocyte ATP pools showed great variability (diversity) among individuals (standard deviation of about 30-40% of mean at baseline). It was discovered that erythrocyte baseline ATP pool sizes are unique to each individual and that they fall within a narrow range in each individual. At the end of an 8 h continuous intravenous infusion of ATP, intracellular erythrocyte ATP pools were increased in the range of 40-60% and extracellular ATP declined from elevated levels achieved at the beginning and middle of the infusion, to baseline levels. The ability of erythrocytes to sequester exogenously administered ATP to this degree, after its initial conversion to adenosine in the blood plasma is unexpected, considering that some of the adenosine is likely to have been degraded by in vivo catabolic activities or taken up by organs. The data suggest that administration of ATP by short-term intravenous infusions, of up to 4 h, may be a favorable way for elevating extracellular ATP pools. A large fraction of the total exogenously administered ATP is sequestered into the intracellular compartments of the erythrocytes after an 8 h intravenous infusion. Erythrocytes loaded with ATP are known to release their ATP pools by the application of previously established agents or conditions applied locally or globally to circulating erythrocytes. Rapid degradation of intravenously administered ATP to adenosine and subsequent accumulation of ATP inside erythrocytes indicate the existence of very effective mechanisms for uptake of adenosine from blood plasma. These in vivo studies offer an understanding as to how both adenosine and ATP can act as purinergic transmission signals. ATP levels in blood are always accompanied by adenosine formed by catabolism of ATP. The continuous uptake of adenosine enables both to act in transmission of sometimes opposite functions.

In vitro evaluation of adenosine 5'-monophosphate as an imaging agent of tumor metabolism.

UNLABELLED: Adenosine appears to play an important role in tumor growth and metastasis. Synthesized (11)C-adenosine 5'-monophosphate (AMP) has recently been reported as a potential tumor-imaging radiotracer. METHODS: A variety of human tumor cell lines (SKOV-3, SCC-15, U251, U87, Raji, and Daudi) were incubated with 3.7 kBq (0.1 microCi) of [2-(3)H]AMP ((3)H-AMP), [5,6-(3)H]FDG ((3)H-FDG), or [2-(3)H]adenosine ((3)H-adenosine) in low-physiologic-glucose serum-free medium. Selected cells were exposed to caffeine, dipyridamole, adenosine 5'-(alpha,beta-methylene)diphosphate (APCP), or unlabeled adenosine before exposure to the radiotracer. R-phycoerythrin-conjugated mouse antihuman monoclonal antibody to human CD73 was used for immunophenotyping. High-performance liquid chromatography was used to characterize the intracellular metabolites of (3)H-AMP after intracellular uptake. RESULTS: Intracellular uptake of (3)H-AMP was significant-10 to 100 times the uptake of (3)H-FDG, depending on the particular tumor cell line. Preexposure of SKOV-3 cells to caffeine, a competitive inhibitor of adenosine receptors, did not affect cellular uptake of (3)H-AMP. However, preexposure of SKOV-3 cells to dipyridamole, an equilibrative nucleoside transporter inhibitor; APCP, a CD73 (ecto-5'-nucleotidase) inhibitor; or cold adenosine significantly inhibited cellular uptake of (3)H-AMP. SKOV-3 uptake of (3)H-adenosine was inhibited by dipyridamole but not APCP. U251 uptake of (3)H-AMP was significantly inhibited by dipyridamole and APCP. U87 uptake of (3)H-AMP was only partially inhibited by dipyridamole and APCP. However, Raji and Daudi cells had significantly lower uptake of (3)H-AMP than of (3)H-FDG but had significantly increased uptake of (3)H-adenosine, which was inhibited by dipyridamole. Raji and Daudi cells were negative, but the SKOV-3 cells positive, for CD73 cell-surface expression. (3)H-Adenosine metabolites were persistently retained after influx into the cell, predominantly as (3)H-adenosine triphosphate and (3)H-adenosine diphosphate. CONCLUSION: Cancer cell lines evaluated in vitro had significantly elevated uptake of radiolabeled AMP, on the order of 10- to 100-fold, in comparison to radiolabeled FDG. The mechanism of intracellular uptake depends predominantly on equilibrative nucleoside transporters after conversion of AMP to adenosine by CD73 in SKOV-3, SCC-15, and U251 cells. Intracellular retention is due to phosphorylation to adenosine triphosphate and adenosine diphosphate. Raji and Daudi cells have low uptake of radiolabeled AMP because of a lack of CD73 expression. This in vitro evaluation using (3)H-AMP with tumor cell lines supports the potential of (11)C-AMP for use in targeting the nucleoside transport pathway in PET imaging of tumors.

Synthesis and biodistribution of [11C]adenosine 5'-monophosphate ([11C]AMP).

PURPOSE: Imaging purine receptors and adenylate biodistribution in vivo may be of clinical importance not only for the investigation of normal adenylate metabolism but also in pathological conditions where adenylate uptake and/or release from certain tissues and organs may be altered, such as some types of cancer. In order to develop a tracer for positron emission tomography (PET) that would not be subject to loss of its radioisotope, adenosine 5'-monophosphate (AMP) was intrinsically labeled at the C-8 position with carbon-11. PROCEDURES: [11C]AMP was synthesized by reacting 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamidine-5'-phosphate with [11C]formaldehyde. The metabolism of [11C]AMP in human blood was determined in vitro both in the presence and absence of dipyridamole. The ex vivo biodistribution of [11C]AMP and its in vivo dosimetry were determined in normal mice. The effect of dipyridamole on the distribution of [11C]AMP in mice was also determined. RESULTS: [11C]AMP was reliably synthesized in 34 minutes (n = 7) with an average radiochemical yield of 2.4% and an average specific activity of 90.10 GBq/micromol (2435 mCi/micromol) at end of synthesis. In normal mice, the highest uptake of [11C]AMP was in the lungs, blood, and heart. The ex vivo mouse experiments showed that the uptake of 11C radiotracer in the lungs at 60 minutes postinjection was significantly lower for dipyridamole-treated animals than controls. Dosimetry showed that the critical organs for radiation dose burden are kidneys and bladder. CONCLUSIONS: Treatment with dipyridamole blocked the red blood cell uptake of extracellular adenosine and therefore its subsequent intracellular conversion to ATP. The biodistribution studies indicate that the tracer has substantial accumulation in the kidneys, lungs, heart, and blood. [11C]AMP is promising as a PET-imaging agent to trace adenylate biology in vivo.

Pentoxifylline in the treatment of radiation-induced fibrosis.

PURPOSE: Fibrotic sequelae remain the most important dose-limiting toxicity of radiation therapy to soft tissue. Functionally, this is reflected in loss of range of motion and muscle strength and the development of limb edema and pain. Tumor necrosis factor alpha and fibroblast growth factor 2 (FGF2), which are abnormally elevated in irradiated tissues, may mediate radiation fibrovascular injury. PATIENTS AND METHODS: In an open label drug trial, we studied the effects of pentoxifylline (400 mg orally tid for 8 weeks) on 30 patients who displayed late, radiation-induced fibrosis at 1 to 29 years posttreatment (40 to 84 Gy). The primary outcome measurement was change in physical impairments thought to be secondary to radiation, including active and passive range of motion (AROM and PROM), muscle strength, limb edema, and pain. Plasma levels of cytokines (tumor necrosis factor alpha and FGF2) also were measured. Twenty-seven patients completed baseline and 8-week assessments, and 24 patients completed baseline, 8-week, and 16-week assessments. RESULTS: After 8 weeks of pentoxifylline intervention, 20 of 23 patients with impaired AROM and 19 of 22 with impaired PROM improved; 11 of 19 patients with muscle weakness showed improved motor strength; five of seven patients with edema had decreased limb girth; and nine of 20 patients had decreased pain. Pretreatment FGF2 levels dropped from an average of 44.9 pg/mL to 24.0 pg/mL after 8 weeks of treatment. CONCLUSION: Patients receiving pentoxifylline demonstrated improved AROM, PROM, and muscle strength and decreased limb edema and pain. Reversal of these delayed radiation effects was associated with a decrease in circulating FGF2.

Effects of oral ATP supplementation on anaerobic power and muscular strength.

PURPOSE: We examined 14 d of oral adenosine 5'-triphosphate (ATP) supplementation on indices of anaerobic capacity and muscular strength. METHODS: Twenty-seven healthy males successfully completed the trial, after randomly receiving in a double-blind manner an oral dose of low dose (150 mg) or high dose (225 mg) ATP, or matched placebo. To improve absorption characteristics, the ATP was enterically coated. Total blood ATP (whole blood and plasma ATP) concentrations, two Wingate anaerobic power tests (30 s), and muscular strength (1RM and three sets of repetitions to fatigue at 70% of 1RM) were measured under three conditions: (i) baseline; (ii) acutely (7d later, no prior supplementation and 75 min after ATP ingestion); and (iii) after 14 d of daily ingestion (post). RESULTS: Statistical analyses showed no significant between or within group treatment effects for whole blood ATP or plasma ATP concentrations for any treatment condition. We also did not observe any treatment effects for any Wingate testing parameter including peak PO, total work, average PO for 30 s, or post-Wingate lactate accumulation. Overall, we observed no significant between group treatment effects for any muscular strength parameter. We did observe several within group differences for the group ingesting the high ATP dosage including 1RM (6.6%; P < 0.04) and repetitions to fatigue during set 1 of posttesting (18.5%; P < 0.007) and total lifting volume at post (22%; P < 0.003). CONCLUSIONS: We conclude that enterically coated oral ATP supplementation may provide small ergogenic effects on muscular strength under some treatment conditions.

Cystic fibrosis transmembrane conductance regulator in human and mouse red blood cell membranes and its interaction with ecto-apyrase.

Elevated blood ATP and increased red blood cell (RBC) ATP transport is associated with cystic fibrosis (CF). In this report, we demonstrate the presence of the wild-type and the DeltaF508 mutant form of the CF transmembrane conductance regulator protein in RBC membranes and its putative interaction with ecto-apyrase, an ATP hydrolyzing enzyme also present in the RBC membrane. RBC membranes of control and DeltaF508 individuals and of wild-type and CF transmembrane conductance regulator-knockout mice were examined by immunoblot using several antibodies directed against different epitopes of this protein. These experiments indicated that human RBC membranes contain comparable amounts of the wild-type CF transmembrane conductance regulator protein and the DeltaF508 mutant form of the protein, respectively. CF transmembrane conductance regulator protein was also detected in wild-type mouse RBC membranes but not in the gene knockout mouse RBC membranes. Antibodies directed against ecto-apyrase co-immunoprecipitated CF transmembrane conductance regulator protein of human RBC membranes indicating a physical interaction between these two membrane proteins consistent with ATP transport and extracellular hydrolysis. We conclude that RBCs are a significant repository of CF transmembrane conductance regulator protein and should provide a novel system for evaluating its expression and function.