When comparing and interpreting results from studies based on animal-models versus those from human subjects, researchers should consider both differences in metabolic rates and biochemical pathways that exist between species. While safety is of paramount importance, not accounting for such differences may also potentially lead one not to consider developing a drug based on phenomena observed in animal models that do not apply to humans.

One notable difference relates to Kleiber’s Law, which states that an organism’s resting energy expenditure (REE) relates to its mass (M) as REE∝M^3/4 (Kleiber, 1932; Brody, 1945). Thus, REE per-unit-mass (REE/M) for small animals is much greater than that of large animals; REE/M is about eight times greater in mice (196 kcal/kg per day) than in humans (24.8 kcal/kg per day) (Wang et al., 2012). Commensurate with a smaller animal’s need to generate energy and therefore resynthesize ATP at a much faster rate, other studies found that excretion of purine degradation products was about seven-times higher in dogs and 40-times higher in rats than in humans (Hitchings, 1966).

Another notable difference in purine metabolism relates to the different end-products of purine degradation. Guanosine and adenosine nucleotide degradation pathways converge on X, which is then degraded in most mammals to slightly soluble UA by XOR and readily soluble allantoin by urate oxidase (UOX) (Johnson et al., 2009). However, a string of UOX genetic mutations in the ape-lineage finally led to its pseudogenization in humans (Kratzer et al., 2014), meaning that the terminal end-product of purine degradation is different in humans (UA) and typical animal models (allantoin). At least partly due to their increased metabolic rate, UOX knockout in mice results in extreme hyperuricemia and is highly lethal (Wu et al., 1994), while it is generally benign in humans (who are all UOX knockouts). In real-world terms, this is an example of the importance of considering human/animal-model differences, because drugs used to treat hyperuricemia such as allopurinol and febuxostat resulted in renal calculi formation in mice and rats but were not expected to cause such problems in humans (Horiuchi et al., 1999).

Taken together, these observations and results suggest that both similarities and differences between biochemical and metabolic pathways need to be considered when using animal models to extrapolate about biologic and clinical phenomena in humans.

Xanthine oxidoreductase is a homodimeric metallo-flavoprotein that has in its N-terminal region two iron-sulfur redox centers, at its C-terminal domain a catalytic center with the molybdenum cofactor molybdopterin (Moco), and the intervening region a flavin-adenine dinucleotide (FAD) binding domain (Battelli et al., 2014). Research almost 100 years ago measured XOR activity in cow milk, and showed in various rat tissues that it had highest activity in liver and spleen, intermediate activity in kidney and lungs, and little to no activity in muscle (Morgan et al., 1922; Massey and Harris, 1997).

Purine molecules form the scaffold for the high-energy phosphates such as ATP and GTP, and they can be either produced de novo or salvaged from nucleobases or nucleosides that have either been previously synthesized and undergone partial degradation or obtained from the diet (Ipata et al., 2011). However, since de novo synthesis expends energy in the form of 7 high-energy phosphate bonds from 6 ATP molecules to regenerate IMP (Figure 1; Garrett and Grisham, 2016), energy-intensive tissues such as skeletal muscle, heart muscle cells, and brain neurons extensively use salvage pathways to maintain their purine levels (Manfredi and Holmes, 1985; Ipata, 2011). In that process, three purine nucleobases can be salvaged by two different enzymes, with adenine phosphoribosyltransferase (APRT) converting adenine (Ade) to AMP, or hypoxanthine-guanine phosphoribosyltransferase (HGPRT; gene HPRT1) converting Guanine (Gua) and Hx to GMP and IMP, respectively. After the salvage of Hx, IMP may then be converted by Adenylosuccinate synthetase (ADSS; genes ADSS and ADSSL1) to S-AMP (adenylosuccinate) which is then converted by Adenylosuccinate lyase (ADSL) to AMP.

That process of purine salvage and its importance was proposed over 40 years ago, and analyses at the time showed that a substantial fraction (∼95%) of the body’s production and intake of purines is salvaged and recycled from Hx back into nucleotides (Murray et al., 1970; Murray, 1971). Analyses of purine metabolism showed that plasma and urine purine levels varied with different levels of physical activity, and Harkness et al. (1983) observed that acute muscular exercise resulted in dramatically increased plasma Hx levels, and from that, they proposed that the release of Hx and its transport between tissues could be considered as a “circulating hypoxanthine pool” (Harkness et al., 1983). Later, analyses indicated that the total purine loss (i.e., excreted in 24 h urine samples) increased with the number of severe acute exercise sessions (Stathis et al., 1999), and Hellsten et al. (1998) showed that after intense exercise, Hx and UA in the blood increased during recovery, and that while salvage pathways recovered IMP still present in the muscle into ATP, Hx that had been released into the blood appeared not to be salvaged, but rather, Hx was degraded into UA by the liver, returned to the circulation, and then taken up by the muscle tissue. A later study by Stathis et al. (2005) investigated whether treatment with an XOR inhibitor (allopurinol) would attenuate total purine output and allow for greater Hx salvage by the muscle after intense exercise, but allopurinol administration was found to actually increase total purine loss. That study also illustrated that Ino and its metabolite Hx can be readily transported out of muscle cells once IMP is de-phosphorylated by 5′-nucleotidase, and that the increased concentrations of Ino and Hx in the blood may then be readily removed into the urine by the kidneys. The high-transfer potential of Ino and Hx out of exercised muscle likely reflects the action of equilibrative nucleoside transporters ENT1 (SLC29A1) and ENT2 (SLC29A2) that passively move nucleosides and nucleobases out of cellular compartments with high metabolite concentrations (Boswell-Casteel and Hays, 2017).

Much of our early knowledge on the existence of purine salvage originated from research on patients with Lesch-Nyhan’s Disease (LND), which results in “mental retardation, spastic cerebral palsy, choreoathetosis, uric acid urinary stones, and self-destructive biting of fingers and lips” (Hamosh, 2017). LND is caused by genetic mutations in the HPRT1 gene that result in a deficiency in HGPRT enzyme function (Seegmiller et al., 1967; Nguyen and Nyhan, 2016). Thus, LND patients are not able to salvage Gua and Hx to GMP and IMP, respectively, and they display increased levels of purine metabolites including Hx, UA, X, and Ino in the blood (Jiménez et al., 1989a). Analysis of the distribution of purine metabolites in the plasma and urine through use of radioactive tracers suggested that purine salvage was a normal mechanism for Hx reutilization in humans (Edwards et al., 1979), and other researchers showed that purines produced in the liver were taken up by erythrocytes and transported to other tissues (Pritchard et al., 1975). Such results, taken together with the severe neurological symptoms that accompany the disease played a key role in showing the importance of purine salvage pathways in normal functioning of the brain.

Although ENT1 and ENT2 have been proposed as the transporters for Hx salvage, their respective K_m values for Hx are quite high (ENT1 K_m = 6.0 ± 0.5 mM; ENT2 K_m = 1.5 ± 0.2 mM) compared to typical plasma Hx concentrations (∼0.46 μM; see Section 2.8, below) (Yao et al., 2011), suggesting that some other more efficient mechanism existed for the transport of Hx across membranes. Thus, Bone et al. (2010, 2015) identified the existence of a nucleobase transporter in studies of Hx transport in cardiac microvascular endothelial cells (MVEC) (Bone and Hammond, 2007) and in MVEC from ENT1-/- mice). Although unable to identify the actual gene or protein involved, they termed the putative transporter as equilibrative nucleobase transporter 1 (ENBT1). That name was later used by Furukawa et al. (2015) who identified solute carrier family 43 member 3 (SLC43A3) as able to uptake nucleobases such as adenine, guanine, and hypoxanthine, but not nucleosides. Interestingly, they also showed that nucleobase import was greater when cells expressed both SLC43A3 and APRT (for adenine import) or HGPRT (for guanine import), suggesting that the nucleobase transporter and salvage enzyme work together in a cooperative fashion, with the final imported product being the nucleotides.

Interestingly, an earlier study by Wallgard et al. (2008) had looked for genes that were differentially expressed in microvascular endothelial cells, and they found SLC43A3 differentially enriched in brain microvessels versus rest-of-brain tissue. In a review of FANTOM5, we found that all of twenty-one human endothelial cell samples expressed SLC43A3. As Furukawa had found that the K_m for Hx was 1.32 μM, which is close to reported normal Hx plasma concentrations, these results suggest that SLC43A3 functions in the microvasculature of various tissues to facilitate purine uptake and salvage. Interestingly, all FANTOM5 skeletal muscle samples also expressed the gene, suggesting a role for nucleobase import and salvage despite the contradictory results from research involving intense muscular exercise.

If that purine salvage process fails to salvage Hx back into high-energy phosphate molecules, and instead, XOR degrades it to X, then X can only be further degraded by XOR to UA (Figure 1). The degradation process may take place within cells if they express XOR or following excretion of Hx from a cell when it traverses organs with XOR activity such as the liver or endothelium. Consequently, processes maintaining cellular energy charge may degrade the molecular scaffold for ATP into a form (UA) that cannot be salvaged back into energy storing molecules. As described above, after degradation to UA, new purine molecules would need to be salvaged from dietary intake or resynthesized de novo at an energy expense of 7 high-energy phosphate bonds from 6 ATP molecules to regenerate IMP (Figure 1; Garrett and Grisham, 2016).

Previous results suggest that serum UA is a biomarker of ATP consumption, since rapid ATP consumption can lead to purine degradation and an increased rate of UA production in humans. For example, intense muscular exercise (Hellsten et al., 1999; Stathis et al., 1994, 1999), fructose challenge (Budillon et al., 1992; Donderski et al., 2015), alcohol intake (Lieber, 1965; Schmidt et al., 2013), and high brain activity (Salvadore et al., 2010; Goodman et al., 2016) can lead to energy crisis and resultant hyperuricemia. In addition, vascular regions undergoing ischemia-reperfusion may produce increased purine degradation products; a sign that a local energy crisis has occurred. For example, one study investigated the difference in the concentrations of adenine nucleotide degradation products between the aortic root (Ao) and the coronary sinus (Cs), before, during, and after cross-clamping of the aorta during cardiopulmonary bypass (Lazzarino et al., 1994). The authors found that pre-clamping Cs blood was slightly increased for Ado, Ino, Hx,and X (< 1 μmol/L), while UA was increased > 10 μmol/L, showing that XOR was present within the coronary vasculature. The Cs-Ao difference for purine degradation products increased 2–3 fold during the ischemic period, and decreased afterward. In addition, malondialdehyde, a marker of oxidative stress, significantly increased while the aorta was clamped, but there was no apparent correlation with post-operative outcomes, suggesting that oxidative stress in such pathologic situations may not be as important as is often thought.

In contrast, UA production is low in infirm aged people (Beberashvili et al., 2015, 2016), those with low nutrition (Beberashvili et al., 2016), and patients with certain neurodegenerative disorders (Rinaldi et al., 2003; de Lau et al., 2005). For multiple sclerosis (MS), decreased plasma/serum UA levels were observed in multiple studies (Druloviæ et al., 2001; Sotgiu et al., 2002; Liu et al., 2012; Moccia et al., 2015), but some studies have also observed that UA levels were increased compared to control subjects (Amorini et al., 2009; Tavazzi et al., 2011). Tavazzi et al. (2011) identified relapsing-remitting MS patients as having lower UA levels compared to secondary progressive or primary progressive MS patients, and the authors have described serum purine metabolite concentrations as related to an imbalance in energy production (Lazzarino et al., 2017).

Evidence suggests that inhibition of purine degradation leads to alterations of bioenergetic pathways and conserved ATP levels. Data that supports this conclusion is found in genetic deficiencies of the AMPD gene family, as well as from clinical and experimental analyses in various organ systems. AMPD enzymes catalyze the conversion of AMP to IMP (Figure 1), and expression of the three AMPD genes, AMPD1, AMPD2, and AMPD3, are differently expressed in various organs and in various types of cells (Morisaki et al., 1990; Mahnke-Zizelman and Sabina, 1992).

AMPD1 is very strongly expressed in skeletal muscle and diaphragm (Morisaki et al., 1990), and it has been examined extensively for the impact of a non-sense mutation that is common in European ancestry populations but rare in most other world-wide population samples (rs17602729; Gln45Ter; C34T; AF_T-EUR = 0.123 in 1000 Genomes)⁵ Originally, deficiency in AMPD1 due to rs17602729 homozygosity (TT) was identified as a putative cause of myopathy (Fishbein et al., 1978). However, greater than 1% of European ancestry populations are AMPD1 deficient, and many of those subjects are asymptomatic, which suggests a heterogeneity of its effects or that other factors might be involved (Verzijl et al., 1998; Sabina and Holmes, 2001; Hanisch et al., 2008). Studies of elite athletes found significantly less C34T carriers compared to controls, with more skewed genotype frequencies in sprint/power athletes versus endurance and mixed-event athletes, and there appeared to be a deficit of TT individuals (Rubio et al., 2005; Muniesa et al., 2010; Tsianos et al., 2010; Ciȩszczyk et al., 2011, 2012; Ginevičienė et al., 2014; Grealy et al., 2015). However, endurance athletes who had reached the elite level who were heterozygous appeared to generally perform comparably to CC individuals (Rubio et al., 2005; Tsianos et al., 2010). Among various exercise studies of normal and AMPD1 deficient subjects (Norman et al., 2001; Tarnopolsky et al., 2001; De Ruiter et al., 2002; Fischer et al., 2007; Norman et al., 2008), the study with the largest sample-size (n_CC = 89; n_CT = 38; n_TT = 12) (Fischer et al., 2007), which performed a short-term high-intensity Wingate cycling test, found that TT subjects exhibited decreased mean-power output and increased fatigue. However, compared to CC and CT individuals, they did not experience depletion of ATP at the end of the test (Norman et al., 2001). In heart failure patients, genetic AMPD1 deficient subjects exhibited decreased systolic blood pressure (de Groote et al., 2006; Safranow et al., 2009; Tousoulis et al., 2014; Feng et al., 2017), an increase in cardiac output (left ventricular ejection fraction) (Gastmann et al., 2004; Collins et al., 2006; Safranow et al., 2009; Feng et al., 2017), and improved heart failure prognosis (Loh et al., 1999; Gastmann et al., 2004). Interestingly, AMPD1 was also reported as a putative target of the diabetes drug metformin (Cheng et al., 2014), and AMPD1 deficient individuals have been reported to have decreased rates of diabetes (Safranow et al., 2009).

AMPD2 is most prominently expressed in non-skeletal muscle tissues, including brain, liver, and thymus (Morisaki et al., 1990). Genetic AMPD2 deficiency leads to increased ATP but a decrease of GTP and has been reported to lead to rare central nervous system (CNS) disorders (Akizu et al., 2013; Novarino et al., 2014).

AMPD3 is most strongly expressed in erythrocytes but is also found in other tissues, including skeletal muscle. Genetic AMPD3 deficiency has been observed, and it has been reported to lead to a 1.5 fold increase of ATP in erythrocytes without any health problem (Ogasawara et al., 1987; Yamada et al., 1994). Studies of AMPD3 deficient mice have also reported increased ATP levels (Cheng et al., 2012; Daniels et al., 2013; O’Brien et al., 2015).

In addition to genetic deficiencies of the AMPD isozymes, experimental studies have shown that perturbation of certain pathways can modulate cellular energy through AMPD. For instance, under conditions that mimic oxidative stress in studies of erythrocyte energy metabolism, Tavazzi et al. (2000, 2001) found that AMPD activity increased when incubated with H₂O₂ or NaNO₂). Those studies suggest that changes to the local erythrocyte environment may result in deficient energy states due to purine loss. Another study by the same group investigated the effect of angiotensin II (AII) on matrix metalloproteinase activity (MMP) in the canine heart. AII administration increased MMP activity, as well as chamber diastolic stiffening and MMP activity, and decreased tissue bioenergetics, while treatment with an MMP inhibitor was shown to decrease the effects of AII and reduce purine loss, likely through inhibition of AMPD activity (Paolocci et al., 2006).

As noted above, heart, skeletal muscle, and brain are among the organs requiring the largest amounts of energy and that are typical targets of mitochondrial diseases, and interestingly, recent human and animal studies have shown that XOR inhibitors (XOIs) such as allopurinol and febuxostat are useful for the treatment of disorders of heart, skeletal muscle and brain.

Within the various reports on XOIs′ beneficial effects, several hypotheses have been raised. Namely, the mechanism of action is likely related to: (1) UA reduction, (2) reactive oxygen species (ROS) suppression, or (3) ATP enhancement.

Evidence suggests that XOIs′ UA lowering effect is unlikely to be the mechanism of those beneficial effects. In animals such as mice and rats, serum UA levels are very low even without XOI treatment (Watanabe et al., 2014), and therefore, the benefits of XOR inhibition that were observed in animal models are unlikely to be due to its further lowering. Also, of importance to brain-related studies, CSF UA concentrations are very low compared to those in blood, with human CSF levels about 5% of those seen in plasma samples ([UA_CSF]∼12.8 μM; [UA_Plasma]∼250 μM) (Harkness, 1988; Jiménez et al., 1989b). Those low levels are related to two things. First, as introduced above, XOR expression in brain tissue is very low, with human and mice brains having 0.8% and 0.44% of levels seen in the highest XOR expressing tissues (Supplementary Tables S1–S4). Second, the blood-brain barrier (BBB) and blood-cerebral spinal fluid barrier have been reported to be only weakly permeable to UA (Jiménez et al., 1989a; Redzic et al., 2001). Considering that evidence, UA reduction in serum is unlikely to affect the brain.

Although many reports hypothesized that the suppression of ROS is the mechanism of the beneficial effects of XOIs, that possibility also appears quite unlikely, since while heart, skeletal muscle and brain are among the organs that consume the largest amounts of energy, they are also among those with the lowest amounts of XOR in humans (Figure 2A; Linder et al., 1999), with XOR activity mostly limited to the vascular endothelium of those tissues (Kelley, 2015). Also, recent reports have shown that inhibition of ROS by scavengers is often harmful because minimal levels of ROS are useful for signal transduction and cancer inhibition (Diebold and Chandel, 2016; Scialò et al., 2016; Quijano et al., 2016). The high ATP consumption that occurs in those organs should also coincide with high ROS production, since ROS is produced along with the consumption of oxygen. However, ROS are produced mostly in mitochondria, and based on a rough calculation of the daily ratio of moles UA excreted to moles O₂ consumed (UA_g.excr = 0.75 g, UA_mole.excr∼2.97 × 10^-3 moles; 22 moles O₂ per day) (Ng et al., 1984; Engelberg, 1996), oxygen consumption by mitochondria is at least 5,000 fold higher than that consumed by XOR. Thus, ROS related damage is more likely to stem from mitochondria than from XOR.

Therefore, most of the ROS produced would stem from mitochondria and thereby can never be suppressed by XOIs. Of course, this may suggest that mitochondria may be damaged by ROS and that that damage leads to ATP depletion. In fact, the association of ROS with various diseases including neurodegenerative disorders may be through the damage of mitochondria by excessive ROS. However, that does not support inhibition of ROS production as the reason for the beneficial effects of XOR inhibition that has been observed in previous studies.

Based on those observations, it is unlikely that ROS inhibition is the reason for XOIs′ beneficial effects. Rather, ATP enhancement through enhanced purine salvage is the more likely mechanism. Support for ATP enhancement as the mechanism can be found in experimental studies using in vitro and animal models as well as human clinical studies.

One such study in the early 2000s of allopurinol’s impact on mitochondrial function examined rat livers under ischemic/hypoxic conditions and found that decreases in ATP levels after ischemia/reperfusion were attenuated with allopurinol treatment (Jeon et al., 2001). Another study around the same time-period examined prepared rat hearts under hypoxic conditions, and the authors reported that both ATP and total adenine nucleotide pools were enhanced after allopurinol administration (Khatib et al., 2001). Later, a report on canines examined the impact of XOIs on cardiac bioenergetics under conditions simulating myocardial ischemia, with subjects examined at rest and then with/without XOI under either basal cardiac work (BCW) or high cardiac work (HCW) after catecholamine administration (Lee et al., 2011). Without XOI, the ratio of PCr/ATP and ADP levels were unchanged at BCW, but the PCr/ATP ratio decreased and levels of ADP increased at HCW. In contrast, with XOI administration, the ratio of PCr/ATP increased and levels of ADP decreased at BCW, while at HCW the increase in ADP levels was attenuated compared to no XOI. Similarly, a study of non-ischemic cardiomyopathy patients showed using ³¹P magnetic resonance spectroscopy (31P-MRS) that allopurinol increased the flux through creatine kinase (CK) and increased the rate of ATP synthesis as well as decreased ADP levels (Hirsch et al., 2012). In a neurological study of hypoxia/ischemia in piglets, authors reported that allopurinol pre-treatment attenuated a decrease in the PCr/Pi ratio, which is another measure of cellular energy status (Peeters-Scholte et al., 2003). Further, a study of potential causes of MS that used a mouse model of experimental autoimmune encephalomyelitis (EAE) reported that XO played a role in mechanisms leading to demyelination, and that EAE progression was reduced with XOI treatment (Honorat et al., 2013). A follow-up study examined NeuroA2 cells treated with rotenone, which causes dysfunction of mitochondrial electron transport and reduces ATP levels, and found that XOI administration to rotenone treated in vitro culture increased cellular ATP levels (Honorat et al., 2017).

It has been reported that serum UA is reduced in patients with neurodegenerative disorders such as PD (Havelund et al., 2017; Sakuta et al., 2017; Wen et al., 2017), ALS (Abraham and Drory, 2014; Paganoni et al., 2018; Zhang et al., 2018), and AD (Euser et al., 2009; Lu et al., 2016), and as mentioned above, many researchers consider that these observations represent a direct protective role of UA suppressing ROS in the brain (Kori et al., 2016). However, since XOR is not expressed in the brain, and UA levels in the brain are naturally very low, it is more likely that low UA in neurodegenerative disorder patients reflects physiologic and biochemical changes that result in decreased energy production and low ATP consumption.

Such a hypothesis is supported by FDG-PET studies of the brain, which use the glucose analog FDG (fluoro-deoxy glucose) to assay metabolic activity through regional glucose uptake. That glucose uptake represents some glycolytic but predominantly mitochondrial activity in the brain for production of ATP (Mosconi, 2013). FDG-PET studies have identified increased regions of hypometabolism, and thus reduced ATP consumption, in the brains of patients with certain neurodegenerative diseases such as AD (Johnson et al., 2012; Lin and Rothman, 2014). Recently, a European task-force recommended use of FDG-PET in answering a number of clinical questions including the differential diagnosis of AD and PD from similar pathologies (Nobili et al., 2018).

If ATP consumption in the brain is reduced in such diseases, the UA precursor Hx should also be low. In line with that, a recent meta-analysis found that a number of metabolomic studies of PD patients identified decreases of compounds either related to energy production or purine-related pathways (Havelund et al., 2017). Among the latter, one report found that Hx levels in PD patients’ plasma were decreased by 41% while UA was decreased by 14% (Johansen et al., 2009). In addition to that data, a proteomics study targeting hundreds of proteins for incidental AD in the longitudinal Framingham Offspring Cohort identified elevations of anthranic acid and glutamic acid and reductions of Hx and taurine (Chouraki et al., 2017), and a MALDI-MSI analysis targeted at hundreds of proteins in Alzheimer’s model mice detected elevations of Hx and taurine (Esteve et al., 2017). Although those results are suggestive of a relationship between purine metabolism and the neurological diseases, we should also note that another potential cause of reduced Hx levels could be decreased physical activity that might accompany PD and AD.

It is of interest that both Hx and taurine were found among hundreds of compounds whose concentrations were different between disease and control subjects. However, in the mouse model, both Hx and taurine were increased, while they were decreased in human AD patients. This may reflect a difference as to the pathogenic mechanisms for AD between mice and humans, with a common facet of both being that unfolding and degradation of mis-folded/abnormal proteins such as amyloid or tau by proteasomes carries a high energy cost (Benaroudj et al., 2003; Peth et al., 2013). In the case of the mouse model, excessive production of misfolded proteins requires the expenditure of large amounts of ATP in order to degrade them, which is evidenced by increased purine degradation products such as Hx. In contrast, in humans the decreased levels of degradation products from energy production and purine pathways reflect energy production dysfunctions that result in a reduced capacity to degrade abnormal proteins. Thus, the elevation of Hx in the model mice and its reduction in the human patients can be explained by the different mechanisms by which amyloid beta and tau accumulation occur between model mice and human disease.

The mechanism by which we propose that XOIs enhance ATP is illustrated in Figure 3, which suggests that the blood concentration of Hx plays an important role. Since XOR exists mostly in liver in humans, the inhibition of XOR leads to the elevation of Hx in the liver, and eventually in the blood. As noted above, for each molecule of purine that is lost, seven high-energy phosphate bonds are needed for the de novo synthesis of IMP. Since elevated Hx saves purines for salvage, XOI should lead to the enhancement of ATP. Support for that was seen in animal models, such that when a XOI alone was administered to mice or rats, marked elevation of Hx occurred (Schmidt et al., 2009; Szasz et al., 2013; Kato et al., 2016). Similarly, in humans, allopurinol treatment of cancer patients before chemotherapy treatment was shown to increase Hx levels by about 1.5-fold (Wung and Howell, 1984).

With respect to brain levels of Hx, under normal physiologic conditions the mean concentration of Hx in CSF was between 1.8 and 2.0 μM from two reports (Harkness and Lund, 1983; Amorini et al., 2009). In blood, reported mean plasma/serum levels for Hx have varied widely as 0.46 μM (Wung and Howell, 1980), 1.5μM (Harkness and Lund, 1983), 4 μM (Amorini et al., 2009), and 32.4 μM (Psychogios et al., 2011), with the most representative value coming from the Wung and Howell (1980) report, which showed clearly that measured Hx concentrations in plasma/serum increases with time between blood draw and isolation of the plasma/serum. Based on those values, CSF in non-diseased individuals appears to be about three to 4-fold enriched for Hx compared to plasma. That enrichment within CSF (Harkness and Lund, 1983) presumably relates to the lack of XOR in brain. Since Hx is known to cross the BBB (Jiménez et al., 1989a), excess Hx produced in the CNS would be expected to move to the blood. However, with XOI treatment, blood levels of Hx should become elevated, and the BBB’s permeability to Hx should allow it to move into the CNS. Alternatively, the less favorable transport kinetics should reduce its movement from the CNS to the main circulation. An analogous situation was observed in a previous study of LND patients who are deficient for HGPRT, the enzyme that salvages Hx to IMP, and thus lack a key node of the purine salvage pathway (Figure 1; Jiménez et al., 1989a). Hx in HGPRT(-) patients was elevated 3.9-times and 6.4-times compared to their reported normal plasma and CSF, respectively, and Hx in HGPRT(-) patients’ CSF was 2.3-times more than in plasma ([Hx_Normal-CSF]∼2.7 μM, [Hx_{Normal-plasma}]∼1.9 μM; [Hx_HGPRT(-)-CSF]∼17.2 μM; [Hx_{HGPRT(-)-plasma}]∼7.3 μM). Interestingly, treatment with an XOI (Allopurinol) increased Hx in the plasma and CSF to roughly equal levels ([Hx_{HGPRT(-)-Allop-CSF}]∼35.0 μM; [Hx_{HGPRT(-)-Allop-plasma}]∼38.62 μM), suggesting that as Hx could not be degraded by the liver due to XOR inhibition, its levels built-up in the blood and finally equilibrated with those in CSF.

The reports described above have shown that XOIs can have a beneficial effect on various diseases, but it may be possible to enhance the effect by simultaneous administration of a Hx source. As noted above, when a XOI alone was administered to mice or rats, a marked elevation of Hx occurred, but mice and rats produce over 25 times more UA per unit body weight as compared to humans (Horiuchi et al., 1999; Hosoyamada et al., 2016). Those higher rates of UA production occur because of the higher rate of energy generation and ATP consumption in small versus large mammals. Due to the difference in purine flux within the body, the substantial elevation of Hx in humans that would be needed to increase purine salvage may not be achieved by only inhibiting XOR. Similarly, provision of additional purines alone would not be beneficial, since oral purines will be substantially broken down to UA in the digestive tract or during absorption. Therefore, treatment using a combination of an XOI with additional purines would be expected to increase purine salvage, decrease energy expenditures by reducing de novo purine synthesis, and achieve better enhancement of cellular ATP.

To examine that last question, clinical studies were performed using healthy human subjects. Treatment with high-dose XOI alone produced only a slight increase in levels of Hx and ATP, while treatment with Ino alone did not increase Hx or ATP (Kamatani et al., 2017). In contrast, only the combination of an XOI and Ino was observed to substantially increase blood levels of Hx and ATP in the human subjects. In an additional study, it was observed that Ino alone could enhance ATP in human erythrocytes incubated in saline, likely due to the lack of XOR in erythrocytes (Kamatani et al., 2018).

Based on those results, Kamatani et al. (2019) performed a small clinical study of the combined use of an XOI and Ino was performed in two mitochondrial disease patients. After treatment, brain natriuretic peptide decreased by 31% for one patient with mitochondrial myopathy, and for the other patient, who had mitochondrial diabetes, insulinogenic index was raised 3.1-fold. In addition to that small study, the combination therapy is currently under evaluation in a mid-sized study of 30 PD patients.

Therapy combining an XOI and Ino is similar to the treatment of deficiency for carbamoyl-phosphate synthetase 2 (CAD). Through treatment with oral uridine, CNS symptoms were dramatically improved (Koch et al., 2017).