Part Two Repurposing Drugs For Highly Prevalent Diseases: Pentoxifylline, An Old Drug And A New Opportunity For Diabetic Kidney Disease

PENTOXIFYLLINE IN DKD

An old-new friend

Pentoxifylline [3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1Hpurine-2,6-dione] is a promising anti-inflammatory methylxanthine derivative with hemorheological actions. Pentoxifylline was approved by the United States FDA for the treatment of intermittent claudication resulting from peripheral vascular disease ＞30 years ago [87–89]. This drug decreases blood viscosity, erythrocyte aggregation, erythrocyte rigidity, and platelet aggregation. The improvement in red blood cell flexibility and deformability leads to improved blood flow [89, 90]. The pharmacological properties of pentoxifylline have been frequently revisited, and recent evidence indicates other possible beneficial effects of this old drug [91]. Thus, the repurposing of pentoxifylline has been suggested for treating brain ischemia, non-alcoholic fatty liver diseases, and preserving skeletal muscle function [90].

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The hemorheological properties and their potential to decrease intraglomerular pressure led to an early interest in pentoxifylline as a therapeutic agent in kidney disease. In 1982, Blagosklonnaia et al. [92] presented the first clinical evidence of the kidney protective effects of pentoxifylline. Diabetic patients treated with 300 mg/day of pentoxifylline for 3 weeks improved eGFR and decreased proteinuria. The possible application of pentoxifylline for kidney protection in DKD was recently renewed as studies showed pentoxifylline anti-inflammatory, anti-proliferative and anti-fibrotic effects [93, 94] (Table 1).

A series of five open-label clinical trials conducted between 1999 and 2006 focused on the potential kidney protective effects of pentoxifylline in DKD (Table 1). First, Navarro et al. [95] reported a 42.2 and 59.3% decrease in serum tumor necrosis factor α (TNFα) and proteinuria levels, respectively, in a small group of patients with DM and advanced CKD under pentoxifylline treatment (400 mg/day; 6 months) when compared with a control group. Posteriorly, two open-label RCTs conducted by Aminorroaya et al. [96] and Rodríguez-Morán et al. [97] also reported a decrease in proteinuria in non-hypertensive type 2 DM patients with microalbuminuria comparable with those achieved with ACEI treatment (captopril) after the administration of 400 mg pentoxifylline three times a day (t.i.d.) for 2 (40% in pentoxifylline-group and 38.5% in captopril-group) and 6 months (77.2% in pentoxifylline-group and 76.6% in captopril group), respectively. In a subsequent randomized, open-label trial, Navarro et al. [98] found an additive percentage decrease in proteinuria of 11.2% in those ARB-treated DM patients who also received 1200 mg/day pentoxifylline for 4 months; i.e. patients receiving pentoxifylline. Pentoxifylline treatment also decreased both serum and urinary levels of TNFα, without significant variations in patients exclusively under therapy with ARB. The antiproteinuric effect of pentoxifylline correlated with a decrease in urinary TNFα levels [98]. Finally, a subsequent RCT by Rodríguez-Morán et al. [99] newly reported a decrease in the levels of both high and low molecular weight urinary protein excretion (73.8 and 86.4% decrease, respectively) in non-hypertensive microalbuminuric type 2 DM patients treated with 400 mg pentoxifylline (t.i.d. for 16 weeks) not receiving ACEi or ARB therapy. An RCT published by Badri et al. [100] showed a 56% decrease in proteinuria in a small group of non-diabetic patients with glomerulonephritis with add-on pentoxifylline therapy to the background RAS blockade without affecting eGFR. Other clinical trials with different study designs, drug dosages, and follow-up periods, also examined the kidney protective effects of pentoxifylline with generally inconclusive results. An open-label controlled clinical trial conducted by Diskin et al. [101] did not find any additive antiproteinuric effect of pentoxifylline in diabetic glomerulosclerosis patients with a background of ACEI and ARB therapy after 1 year of follow-up. Important concerns of this study are its non-randomized design, the small number of participants (14 patients), and the use of dual RAS blockade, which has important safety concerns [25, 112]. In a double-blind RCT, Perkins et al. [102] also found no differences in proteinuria in 40 DKD patients with mild to moderate CKD after 1 year of add-on pentoxifylline therapy to RAS blockade. However, they observed deceleration in renal function decline in the group treated with pentoxifylline when compared with the control group, with a mean difference between groups of 6.0 mL/min/1.73 m2, and argued that the proteinuria may not always constitute an optimal surrogate outcome parameter in these studies.

To date, the most important RCT evaluating the kidney protective effects of pentoxifylline in DKD is the Pentoxifylline for Renoprotection in Diabetic Nephropathy (PREDIAN) study, published in 2015 by Navarro Gonzalez et al. [84]. The study comprised 169 types 2 DM patients with CKD stages 3 and 4 and residual albuminuria despite RAS blockade. After 2 years of follow-up, patients randomized to the active group (1200 mg/day of pentoxifylline on top of RAS blockade) presented a decrease in the rate of progression of kidney disease, with an eGFR mean difference between groups of 4.3 mL/min/1.73 m2, accompanied by a 14.9% decrease in proteinuria (increased by 5.7% in the control group). The deceleration in the decline of GFR in the pentoxifylline arm began at month 6 and reached statistical significance after 1 year, suggesting that the therapeutic benefit may only be observed in the long term. Moreover, urine TNFα presented a 10.6% decrease in the pentoxifylline group, with no changes in the control group.

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At present, the identification of the central role of inflammation in the development and progression of CKD and its possible therapeutic targets constitutes an important field of research for nephrologists. The anti-inflammatory actions elicited by pentoxifylline have been related to antialbuminuric effects [93, 113–118]. In this regard, an antiproteinuric or kidney function preservation effect of pentoxifylline has also been found in non-diabetic subjects. Goicoechea et al. [103] reported stabilization of kidney function and a significant decrease in markers of inflammation, such as TNFα, fibrinogen, and high sensitivity C-reactive protein (CRP; a 45.5, 11.1, and 57.4% decrease, respectively) in patients with stage 3 CKD or higher who received pentoxifylline therapy when compared with those exclusively on RAS blockade. Proteinuria did not decrease in the pentoxifylline group, although there was a drop-out and incomplete follow-up rate. Lin et al. [104] found that pentoxifylline on top of ARB background therapy stabilized GFR and decreased proteinuria (−23.9%) in macroalbuminuric CKD stage 3 patients after 1 year of follow-up as compared with ARB monotherapy, for whom proteinuria increased 13.8%. Moreover, pentoxifylline decreased urinary levels of TNFα and monocyte chemoattractant protein 1 (MCP-1) (TNFα: 42.8% versus 18.8% and MCP-1: −28.9% versus 6.2%, for pentoxifylline and control groups, respectively). A decrease in both parameters was directly related to the change in proteinuria in the pentoxifylline group. Chen et al. [105] reported that 800 mg/day pentoxifylline for 6 months decreased proteinuria in 17 patients with primary glomerular diseases [36.5% and 33.9% decrease in spot and 24 h proteinuria (g/g Cr)]. This decrease was associated with a decline in urinary mean percentage decrease of 46% in MCP-1 urinary excretion levels. In a larger study, Chen et al. published a retrospective analysis of a study comprising 661 patients with CKD stages 3–5 treated with pentoxifylline [106]. Again, pentoxifylline on top of RAS blockade had kidney protective effects in the subset of patients with higher levels of proteinuria. A trial conducted by Shu et al. [107] reported a 19.6% decrease in proteinuria in the third month and improved graft survival by the end of the study in non-diabetic renal transplant recipients with chronic allograft nephropathy and microalbuminuria treated with pentoxifylline for at least 6 months.

Two recent meta-analyses reported the effects of pentoxifylline alone or in combination with other treatments in the decrease in proteinuria and the preservation of kidney function in patients with diabetic or non-diabetic CKD. In the first meta-analysis, Leporine et al. [119] concluded that pentoxifylline was effective in decreasing proteinuria compared with control, a benefit that was more evident in patients with type 1 DM, higher proteinuria at baseline, and early renal impairment. They also found an improvement in renal function (eGFR/creatinine clearance) in the long term and in patients with more advanced CKD. In the second meta-analysis, Liu et al. [120] concluded that pentoxifylline in combination with RAS blockade decreases proteinuria and slows down the decline of renal function in patients with CKD stages 3–5.

Finally, an analysis of a nationwide administrative dataset of advanced CKD patients identifying two propensity score matched cohorts (pentoxifylline users and nonusers) reported that the pentoxifylline group was protected from ESKD [121] This was the first evidence of the ability of pentoxifylline in decreasing the risk of ESKD even in patients with advanced CKD.

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Mechanisms of kidney protection by pentoxifylline

Pentoxifylline is a methyl-xanthine derivative with several effects including the non-selective inhibition of phosphodiesterases (PDEs). The balance of intracellular cyclic adenosine- 3,5-monophosphate (cAMP), an important intracellular second signaling messenger, is mainly dependent on the activity of two enzymes: adenylyl cyclase, which plays a major role in the cAMP synthesis and PDEs, which hydrolyze cAMP [122, 123]. Therefore, the inhibition of PDEs by pentoxifylline prevents the degradation of cAMP (Fig. 1). High cAMP levels, in turn, promote protein kinase A (PKA) activation leading to the phosphorylation of diverse effectors followed by inhibition of signaling pathways involved in proteinuria and renal fibrosis [124–126].

PDEs have emerged as promising targets for pharmacological intervention against CKD progression [127–129]. Mammalian cells have 11 gene families (PDE1–PDE11) and each family encompasses 1–4 distinct genes, giving ˃20 genes in mammals encoding ˃60 different PDE isoforms. In vitro, pentoxifylline inhibits PDE3 and/or PDE4 isozymes through a PKA-dependent pathway [124, 126, 130]. Importantly, PDE3 and PDE4 isozymes are mainly expressed in monocytes and neutrophils [131–133], which makes them a therapeutic target in many inflammatory diseases, including asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, psoriasis, nervous system inflammation, and rheumatoid arthritis [133]. Pentoxifylline presents anti-inflammatory properties mediated by the inhibition of PDEs that supports its potential application in the kidney protection of patient with DM [113–118]. In experimental models, pentoxifylline modulates signaling pathways or components triggered by inflammatory cytokines. In vitro, pentoxifylline inhibits endotoxin-induced TNFα synthesis in RAW 264.7 macrophages [108]. Pentoxifylline also inhibited endotoxin-induced TNFα production both in the serum of mice and in cultured adherent peritoneal exudate cells [93]. In a rat model of crescentic glomerulonephritis, pentoxifylline exerts antiinflammatory and immunomodulatory actions through the inhibition of renal TNFα, ICAM-1, RANTES, MCP-1, and OPN, thereby suppressing progressive renal injury [109]. Similarly, pentoxifylline decreases the renal expression of pro-inflammatory cytokines including TNFα and IL6 in streptozotocin- or alloxan-induced diabetic rat models [110, 111], ameliorating renal hypertrophy and sodium retention [110]. Clinical trials evaluating the anti-inflammatory properties of this drug in non-diabetic patients report considerable modulating effects on the production of inflammatory cytokines and adhesion molecules in patients with coronary artery disease and atherosclerosis [134, 135]. Similarly, pentoxifylline decreased TNFα and interferon-gamma Tcell expression in ESKD patients [136].

As discussed above, most RCTs evaluating the renal effects of pentoxifylline in patients with DM have shown kidney protection, evidenced by the decrease in proteinuria and, in some cases, the improvement or preservation of GFR (Fig. 2). Importantly, some RCTs also observed a significant decrease in inflammatory parameters. The antiproteinuric effect of pentoxifylline has been associated with a significant decrease in TNFα levels [95, 99]. Similarly, clinical trials conducted in CKD patients with stage 3 or higher reported stabilization of renal function and decreased circulating levels of TNFα, fibrinogen, and CRP after treatment with pentoxifylline [118] and a decrease in proteinuria and urinary levels of TNFα and MCP1 after 1 year on add-on pentoxifylline to ARB background therapy [104]. The PREDIAN trial [84] evaluated the kidney-protective effects of pentoxifylline in DKD patients under RAS blockade. After 2 years, patients on pentoxifylline presented a decrease in the progression of renal disease that was accompanied by a decrease in proteinuria and urinary levels of TNFα. Two meta-analyses also pointed to the decrease of proinflammatory cytokines production as the most likely explanation for this antiproteinuric effect in DKD patients [137] and concluded that pentoxifylline additively decreases proteinuria and TNFα in DKD patients receiving RAS inhibitors [138].

An unexpected beneficial effect of pentoxifylline in DKD patients could be the stimulation of factors that promote kidney health [139]. The protein Klotho is an important regulator of mineral metabolism mainly expressed in kidney tubular epithelial cells and, to a lesser extent, in parathyroid glands, choroid plexus of the brain, vascular tissue, and peripheral blood cells [140, 141]. Two forms of Klotho can be found: a single-pass transmembrane protein and a soluble form generated from proteolytic cleavage of the extracellular domain of the membrane-bound form [142]. Soluble Klotho is found in the cerebrospinal fluid, urine, and blood, and declines in CKD patients with the progression of the disease. Klotho has anti-aging and kidney-protective effects. Specific epigenetic prevention of Klotho downregulation prevented acute kidney injury in mice [143]. Interestingly, patients with type 2 DM also have lower soluble Klotho levels [144, 145] and kidney Klotho is decreased in biopsies from patients with early stages of DKD [146]. Taken together, these data point to the potential utility of Klotho as an early biomarker of renal impairment in type 2 DM patients and to Klotho downregulation as a driver of DKD progression [147]. Moreover, Klotho has been inversely related to inflammation. Pro-inflammatory cytokines like TNFα and TWEAK (tumor necrosis factor-like weak inducer of apoptosis) inhibit renal Klotho expression in an NF-κB-mediated manner in vivo and in vitro [148–151]. Clinical translation of this observation is supported by a post hoc analysis of the PREDIAN trial [84]. Administration of pentoxifylline to type 2 DM patients with CKD stages 3 and 4 decreased serum and urinary TNFα and increased serum and urinary Klotho levels [85]. Even though the precise mechanisms are unknown, a feasible hypothesis is that the stimulation of Klotho production by pentoxifylline may result from its anti-inflammatory properties, although in cultured tubular cells, pentoxifylline directly prevented the albuminuria-induced downregulation of Klotho expression [85, 152]. Moreover, pentoxifylline increased tubular cell Klotho above baseline levels, suggesting that promoting kidney expression may be one of the mechanisms of kidney protection by pentoxifylline. Moreover, a recently published experimental study, confirmed the positive effect of therapeutic doses of pentoxifylline (10 μg/mL) on Klotho expression in RAW 264.7 cells, showing that this up-regulation is not limited to kidney cells [153].

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CONCLUSIONS AND FUTURE PERSPECTIVES

The global burden of diabetes is predicted to increase dramatically in the coming decades in parallel with the rising of obesity. One of the most important complications of diabetes is DKD, which substantially increases cardiovascular morbidity and mortality, determining a considerable impairment in the quality of life. Indeed, CKD is set to become the fifth global cause of death by 2040 [154]. In a recent large, retrospective cohort study including 65 000 adults with type 2 DM and CKD, a high proportion (10%–17%) of patients presented disease progression over a median follow-up of only 2 years [153]. Previous studies also indicated that DM is a more frequent cause of accelerated progression from CKD to ESKD compared with other predictors including proteinuria, heart failure, anemia, and elevated systolic blood pressure [155].

Even with the widespread use of SGLT2i and GLP-1 receptor agonists, a substantial residual risk of DKD progression remains. Therefore, there is a need to find new therapeutic targets and strategies. Pentoxifylline constitutes a potential repurposed drug for the treatment of DKD. Although the repurposing of pentoxifylline constitutes a promising opportunity in providing low-cost access to a feasible therapy in DKD, various challenges remain. A recent study reported an increased risk of major bleeding events in CKD patients on pentoxifylline treatment [156]. This population carries a higher risk of bleeding due to the presence of platelet dysfunction and anemia, especially in patients with albuminuria [157, 158].

As pointed out by Leporini et al. [119] in a systematic review and meta-analysis aimed at evaluating the benefits of pentoxifylline on renal outcomes in CKD patients, whether this drug may be useful for retarding disease progression remains partly unanswered for definite reasons. One of them is the heterogeneity of the studies concerning sample sizes, doses of pentoxifylline, the population in the study (CKD stage and etiology, proteinuria levels), control groups, and concomitant RAS-blocking treatments. Most RCTs were of low methodological quality and carried out with small sample sizes and only provided short-term data for surrogate kidney function endpoints such as eGFR or serum creatinine, proteinuria, and albuminuria.

Future studies with longer follow-ups, larger sample sizes, and hard clinical outcomes are needed. To our knowledge, at present, two-phase IV clinical trials are evaluating the effects of pentoxifylline in the progression of DKD patients. The Veterans Affairs (VA) PTXRx (NCT03625648) is a double-blind, placebo-controlled, multicentre RCT designed to evaluate the utility of pentoxifylline, when added to usual care, in the decrease in the incidence of ESRD or death in patients with type 2 diabetes with DKD [159]. The enrolment of patients for this study (which is estimated at 2510 participants) began in 2019 and the primary completion date will be at the beginning of 2028. The second clinical trial in the course, PENFOSIDINE (Pentoxifylline Effect in Patients With Diabetic Nephropathy, NCT03664414), began in 2018. PENFOSIDINE included 196 patients with DKD and the objectives included evaluating the antioxidant, anti-inflammatory, and antifibrotic effects of receiving pentoxifylline (400 mg/three times a day) for 2 years. The results of these trials will shed light on the long-term effects of pentoxifylline on various markers of inflammation, oxidative stress, and fibrosis, on surrogate markers of renal function such as a decrease in proteinuria and changes in eGFR, and on hard endpoints such as ESRD and death.

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In any case, an important problem that faces the repurposing of many drugs, including pentoxifylline, is the absence of exclusivity for the industry. Since pentoxifylline was approved for intermittent claudication, no studies were conducted about its utility for other diseases before the approval or before the expiration of the patent. This implies that funding for future research is highly compromised even though current research may demonstrate the efficacy of pentoxifylline in other diseases. Pharmaceutical companies find it less lucrative redirecting resources toward repurposing programs given the lack of standardized regulations ensuring market protection. Moreover, even after marketing approval, the off-label and unlicensed prescription of a generic version of this old drug leaves little or no space for profit for the industry. Therefore, pivotal RCTs in search of a DKD indication for pentoxifylline should be funded by government-supported trial programs.

In addition, a successful new indication of the repurposed drug requires extensive knowledge of the pathogenesis of the target disease, as well as the postulated mechanism of action of the drug. The present data point to the ability of pentoxifylline to slow CKD progression when macroalbuminuria is present, even in advanced stages of DKD with maximized RAS blocker therapy. However, although there is evidence for an anti-inflammatory effect, the precise mechanism for the beneficial effect is not known and may also involve increasing Klotho production. The pathogenic pathways for DKD are poorly understood, as exemplified by the poor therapeutic toolkit and low-grade inflammation is just one of the interrelated contributing mechanisms along with hyperglycemia, altered lipid metabolism, RAS hyperactivation, and increased sympathetic activity. In any case, an in-depth study of the hidden therapeutic potential of pentoxifylline and its repurposing for the DKD new indication offers tremendous hope to decrease residual kidney risk and prevent the growth of the DKD pandemic.

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Javier Donate-Correa1,2,3, María Dolores Sanchez-Niño4, Ainhoa González-Luis1,5, Carla Ferri1,5, Alberto Martín-Olivera1,5, Ernesto Martín-Núñez1,3, Beatriz Fernandez-Fernandez4,6, Víctor G. Tagua1, Carmen Mora-Fernández1,2,3, Alberto Ortiz 4,6, and Juan F. Navarro-González 1,2,3,7,8

1 Unidad de Investigación, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain,

2 GEENDIAB (Grupo Español para el estudio de la Nefropatía Diabética), Sociedad Española de Nefrología, Santander, Spain,

3 RICORS2040 (RD21/0005/0013), Instituto de Salud Carlos III, Madrid, Spain,

4 Departamento de Nefrología e Hipertensión, IIS-Fundación Jiménez Díaz y Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain,

5 Escuela de doctorado, Universidad de La Laguna, La Laguna, Spain,

6 RICORS2040 (RD21/0005/0001), Instituto de Salud Carlos III, Madrid, Spain,

7 Servicio de Nefrología, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain

8 Instituto de Tecnologías Biomédicas, Universidad de La Laguna, Santa Cruz de Tenerife, Spain