Polyethylene Glycol-modified Adenosine Deaminase Research Articles

A2B Adenosine Receptor–Mediated Induction of IL-6 Promotes CKD

Chronic elevation of adenosine, which occurs in the setting of repeated or prolonged tissue injury, can exacerbate cellular dysfunction, suggesting that it may contribute to the pathogenesis of CKD. Here, mice with chronically elevated levels of adenosine, resulting from a deficiency in adenosine deaminase (ADA), developed renal dysfunction and fibrosis. Both the administration of polyethylene glycol-modified ADA to reduce adenosine levels and the inhibition of the A(2B) adenosine receptor (A(2B)R) attenuated renal fibrosis and dysfunction. Furthermore, activation of A(2B)R promoted renal fibrosis in both mice infused with angiotensin II (Ang II) and mice subjected to unilateral ureteral obstruction (UUO). These three mouse models shared a similar profile of profibrotic gene expression in kidney tissue, suggesting that they share similar signaling pathways that lead to renal fibrosis. Finally, both genetic and pharmacologic approaches showed that the inflammatory cytokine IL-6 mediates adenosine-induced renal fibrosis downstream of A(2B)R. Taken together, these data suggest that A(2B)R-mediated induction of IL-6 contributes to renal fibrogenesis and shows potential therapeutic targets for CKD.

Adenosine Deaminase Enzyme Therapy Prevents and Reverses the Heightened Cavernosal Relaxation in Priapism

Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility. The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism. Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol-modified ADA (PEG-ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG-ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation. Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays. We found that lowering adenosine levels in penile tissues by PEG-ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG-ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism. Our studies have identified that PEG-ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation seen in two independent priapic animal models, and suggested its therapeutic possibility in men suffering from priapism.

Detrimental Role of Excess Adenosine-Mediated 2,3-Diphosphoglycerate Induction in Erythrocyte Sickling and Novel Mechanism-Based Therapies.

Abstract 906 Introduction:Sickle cell disease (SCD) is a devastating inherited hemolytic disorder due to a single missense mutation in the β-globin gene resulting in the production of hemoglobin S (HbS). Despite our knowledge of the molecular defect in HbS, we remain unable to control HbS polymerization and erythrocyte sickling under hypoxic conditions, which are central to the pathophysiology of the disease. Methods and Results:Here we report that adenosine, a cellular metabolite that is well known to be induced under hypoxic/ischemic conditions, is increased in the blood circulation of patients with SCD and in SCD transgenic (Tg) mice. To our surprise, we found that adenosine contributes to hypoxia-induced sickling of cultured erythrocytes from humans with SCD. Next, to evaluate the pathogenic role of increased adenosine in erythrocyte signaling in vivo, we took advantage of SCD transgenic (Tg) mice, a well accepted animal model of SCD. Intriguingly, treatment of SCD Tg mice with polyethylene glycol-modified adenosine deaminase (PEG-ADA) enzyme therapy to lower circulating adenosine levels reduced sickling and attenuated multiple tissue damage (including lung, kidney, liver and spleen) seen in SCD Tg mice. These findings revealed a previously unrecognized role for excess adenosine in sickling and the progression to multiple life-threatening complications. Next, by screening erythrocytes for small metabolites that are induced by adenosine and may contribute to sickling, we determined that 2,3-diphosphoglycerate (2,3-DPG), an erythroid specific metabolite known to promote O2 release from Hb, is elevated in erythrocytes of both SCD Tg mice and humans. Using both pharmacological and genetic approaches, we demonstrated that adenosine-mediated 2,3-DPG induction is through A2B receptor signaling and that this signaling pathway is a major contributor to hypoxia-induced erythrocyte sickling. Conclusion and Significance:Overall, we have identified for the first time that elevated adenosine induces 2,3-DPG via A2BR signaling and contributes to sickling in both human and mouse with SCD. Thus, our studies provide strong support for the new concept that adenosine-mediated 2,3-DPG induction is beneficial for normal erythrocytes by promoting O2release from Hb to hypoxic tissues. However, for SCD patients, the beneficial effect of adenosine-mediated 2,3-DPG induction becomes detrimental by inducing erythrocyte sickling due to 2,3-DPG mediated increased O2release and increased deoxyHbS polymerization. Our findings reveal a novel therapeutic possibility to treat and prevent sickling and progression to multiple life-threatening complications by targeting on the adenosine signaling pathway. Disclosures:No relevant conflicts of interest to declare.

Adenosine Signaling, Priapism and Novel Therapies

Priapism is defined as abnormal prolonged penile erection lasting at least for 4 hours occurring without sexual interest. Forty percent of sickle cell disease (SCD) patients display priapism. The disorder is dangerous and urgent given its association with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. Current strategies to manage the disorder are poor due to lack of fundamental understanding of the molecular mechanisms of priapism. Adenosine is a signaling nucleoside that elicits many pathophysiological effects by engaging membrane receptors. Recent evidence shows that adenosine may play an important role in priapism via adenosine receptor. To summarize the recent findings on the importance of adenosine signaling in the pathogenesis of priapism. Evidence in the literature on the association between adenosine signaling and the development of priapism. This article reviews the literature that relates to the contributory role of adenosine signaling in priapism in multiple animal models and humans. Excessive adenosine accumulation in the penis, coupled with increased A(2B)R signaling, contributes to priapism in two independent lines of mutant mice. One is adenosine deaminase (ADA)-deficient mice, the only animal displaying spontaneously prolonged penile erection, and the other is SCD transgenic mice, a well-accepted priapic animal model. Both polyethylene glycol-modified ADA (PEG-ADA) enzyme therapy and A(2B)R antagonists are capable of inhibiting potent corpus cavernosal vascular relaxation associated with priapic-like activity seen in both ADA-deficient mice and SCD transgenic mice, indicating that PEG-ADA enzyme therapy is likely to be a novel therapy for such a dangerous urological disorder. Overall, the research reviewed here raises the intriguing possibility that elevated adenosine signaling contributes to priapism in general and that this signaling pathway represents a potentially important therapeutic target for the treatment of priapism.

Reduced thymic output, increased spontaneous apoptosis and oligoclonal B cells in polyethylene glycol-adenosine deaminase-treated patients

Impairment of purine metabolism due to adenosine deaminase (ADA) deficiency is associated with a severe combined immunodeficiency (SCID). Polyethylene glycol-modified ADA (PEG-ADA) has provided noncurative, life-saving treatment for these patients, but full immune recovery is not achieved with this therapy. Since ADA-SCID is perhaps the most difficult form of SCID to handle clinically, understanding the benefits and limitations of PEG-ADA therapy may be relevant for treatment selection. To this purpose, we analyzed the rate of thymic output, T and B cell repertoires, number of T cell divisions, IFN-gamma and IL-4 production, and the extent of cell death in five ADA-SCID patients following a prolonged period of treatment with PEG-ADA. We found that thymic output was low in these patients. However, their T cell repertoire was heterogeneous, and their T lymphocytes produced cytokines upon activation and responded to mitogen stimulation, although with different kinetics. Furthermore, a high number of peripheral T lymphocytes were committed to apoptosis. Anomalies were also observed in the B cell compartment, with oligoclonal expansions of B cell clonotypes in two patients. Our data indicate that decreased thymic function, B cell oligoclonality, and increased spontaneous apoptosis may be the mechanisms by which the immunodeficiency of ADA-SCID patients persists in spite of treatment with PEG-ADA.

Food allergy in a patient with adenosine deaminase deficiency undergoing enzyme replacement with polyethylene glycol–modified adenosine deaminase

J Allergy Clin Immunol 1998;101:561-2.

PEG-ADA replacement therapy for adenosine deaminase deficiency: an update after 8.5 years

Polyethylene glycol-modified adenosine deaminase (PEG-ADA) has now been used for 8.5 years as enzyme replacement therapy for immunodeficiency due to ADA deficiency. PEG-ADA restores a metabolic environment necessary for recovery of immune function. In most cases, the level of function achieved has been sufficient to protect against opportunistic and life-threatening infections. To date, mortality and morbidity with PEG-ADA have been less than for haploidentical bone marrow transplantation. As a true "orphan drug" used to treat a very small patient population, the cost per patient of PEG-ADA is very high, but it has been well tolerated, free of adverse reactions, and effective as an alternative for patients who lack an HLA-identical marrow donor, but are considered too ill to undergo haploidentical marrow transplantation. Concomitant treatment with PEG-ADA has also permitted investigation of gene therapy to be carried out safely.

IgG antibody response to polyethylene glycol-modified adenosine deaminase in patients with adenosine deaminase deficiency.

Polyethylene glycol (PEG)-modified bovine adenosine deaminase (ADA) is used for replacement therapy of severe combined immunodeficiency disease due to inherited ADA deficiency. We monitored IgG anti-ADA antibody in 17 patients treated by intramuscular injections of PEG-ADA for 1 to greater than 5.5 yr. ELISA-detectable anti-ADA IgG appeared in 10 patients, usually between the third and eighth months of treatment. Anti-ADA levels did not correlate with trough plasma ADA activity, which averaged 1.8-5 times normal blood (erythrocyte) ADA activity, depending on dose (15-60 U/kg per wk). ELISA-detectable anti-ADA antibodies were directed primarily at bovine-specific peptide (rather than PEG-containing) epitopes. Enhanced enzyme clearance, mediated by antibody that directly inhibited native and PEG-modified bovine ADA, and native, but not PEG-modified human ADA, occurred in two patients. In one, tolerance was induced; in the second, twice weekly injections of PEG-ADA compensated for accelerated clearance. We speculate that inhibitory antibodies recognize conserved, relatively PEG-free epitope(s) encompassing the active site, and that in human, but not bovine, ADA a PEG-attachment site "shields" the active site from immune recognition. We conclude that PEG-modification largely prevents the development of high affinity, or high levels of clearing antibodies to bovine ADA, and that PEG-modified human ADA should be further investigated as a possible treatment for ADA deficiency.

Comparison of Red Cell Transfusion and Polyethylene Glycol-Modified Adenosine Deaminase Therapy in an Adenosine Deaminase-Deficient Child: Measurement of Erythrocyte Deoxyadenosine Triphosphate as a Useful Tool

The effect of red cell transfusion and polyethylene glycol-modified adenosine deaminase therapy on biochemical abnormalities, clinical status, and immunologic function in an adenosine deaminase-deficient child was investigated. After red cell transfusions, erythrocyte deoxyadenosine triphosphate (dATP) concentrations decreased about 95% and were closely related to adenosine deaminase activities; deoxyadenosine diphosphate concentrations decreased only approximately 30%. The evolution of dATP levels was also closely related to the improvement in clinical status of the patient. However, immune function was not restored. After polyethylene glycol-modified adenosine deaminase therapy, the concentration of erythrocyte dATP decreased to undetectable levels correlated with an increase of T lymphocyte counts and an increase of lymphocyte responses to mitogens. Immune functions were restored only when dATP levels were below 15 mumols/L. It appears that red cell transfusion therapy is not sufficiently effective to reduce and maintain erythrocyte dATP levels at values compatible with normal immune function. On the contrary, polyethylene glycol-modified adenosine deaminase therapy is a suitable treatment to reduce dATP levels to near undetectable values, allowing the immune function to be restored, dATP measurement is a very useful tool for monitoring and evaluating the degree of efficiency of therapy in adenosine deaminase deficiency.

Adenosine deaminase deficiency with late onset of recurrent infections: Response to treatment with polyethylene glycol-modified adenosine deaminase

We report a 5-year-old girl with adenosine deaminase (ADA) deficiency who was asymptomatic during the first years of life. At 3 years of age, she developed chronic and recurrent sinopulmonary infections, and at 4 1/2 years of age she had one major infection with Streptococcus pneumoniae (bacteremia and septic arthritis of the hip). Immunologic evaluation at 5 years of age revealed persistent lymphopenia, decreased helper-suppressor T cell ratios, and low proliferative responses to mitogens. The IgG, IgM, and IgA levels were normal; the IgG2 level was low normal or below normal. The patient had specific antibodies against toxoids and viral antigens but failed to produce antibodies against Haemophilus influenzae type b and pneumococcal polysaccharides. Although no symptoms of allergy were present, she had persistent eosinophilia and elevated IgE levels. The patient had 0.6% of normal ADA activity in erythrocytes and approximately 1% of normal ADA activity in peripheral blood mononuclear cells. Beginning at 6 years of age, she was treated with weekly injections of polyethylene glycol-modified bovine ADA. This treatment was well tolerated and effectively reversed the biochemical consequence of ADA deficiency. Concomitantly, she improved clinically and her T lymphocyte numbers and blastogenic responses to mitogens in vitro became normal. The late onset of clinical symptoms and relatively benign clinical course in this patient emphasize the need to consider ADA deficiency in a broad spectrum of immunodeficient children.

The availability of purines influences both the number of parasites and the splenocyte levels of purine-metabolizing enzymes in trypanosome-infected mice.

Growth on Trypanosoma musculi in the murine host was limited by the availability of host purines. A portion of the spleen cells of infected mice (many of them granulocytes) displayed high levels of adenosine deaminase (ADA) and purine nucleoside phosphorylase, probably as a compensatory response to extracellular purine deficiency. Injections of adenosine or 2-deoxycoformycin stimulated significant increases in the growth of parasites. 2-Deoxycoformycin treatment also diminished parasite-induced splenomegaly. Treatment of mice with polyethylene glycol-modified ADA, a slowly catabolized form of ADA, had no effect on the course of T. musculi infection, indicating that the parasites can utilize purines other than adenosine. The apparent competition between parasites and host cells for available purines suggests that depletion of extracellular purines should be considered as an approach to treating extracellular trypanosome infections.

Treatment of Adenosine Deaminase Deficiency with Polyethylene Glycol–Modified Adenosine Deaminase

We treated two children who had adenosine deaminase deficiency and severe combined immunodeficiency disease by injecting bovine adenosine deaminase modified by conjugation with polyethylene glycol. The modified enzyme was rapidly absorbed after intramuscular injection and had a half-life in plasma of 48 to 72 hours. Weekly doses of approximately 15 U per kilogram of body weight maintained plasma adenosine deaminase activity at two to three times the level of erythrocyte adenosine deaminase activity in normal subjects. The principal biochemical consequences of adenosine deaminase deficiency were almost completely reversed. In erythrocytes, adenosine nucleotides increased and deoxyadenosine nucleotides decreased to less than 0.5 percent of total adenine nucleotides. The activity of S-adenosylhomocysteine hydrolase, which is inactivated by deoxyadenosine, increased to normal in red cells and nucleated marrow cells. Neither toxic effects nor hypersensitivity reactions were observed. In vitro tests of the cellular immune function of each patient showed marked improvement, along with an increase in circulating T lymphocytes. Clinical improvement was indicated by absence of infection and resumption of weight gain. We conclude that from the standpoints of efficacy, convenience, and safety, polyethylene glycol-modified adenosine deaminase is preferable to red-cell transfusion as a treatment for adenosine deaminase deficiency. Patients with other inherited metabolic diseases in which accumulated metabolites equilibrate with plasma could benefit from treatment with the appropriate polyethylene glycol-modified enzyme.