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Experimental Uridine Therapy for Lipoatrophy
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Written by Jules Levin
Today is nucleoside day, I will report information presented at the Workshop on nucleosides, lipoatrophy, and study data on switching nukes to prevent or improve fat loss. First, at the Lipodystrophy Workshop today Ulrich Walker gave an oral presentation on uridine, a potential therapy for mitochondrial pepletion due to NRTIs and for lipoatrophy. Walker initially presented about uridine as a therapy for lipoatrophy at the Paris Lipoatrophy Workshop last summer. There are two ongoing clinical studies including one within the ACTG looking at uridine as a treatment for lipoatrophy. Below are 4 reports on uridine. One was a letter published in the journal AIDS, 2 are reports on uridine presentations at the 2003 paris Lipodystrophy Workshop, and immediately below is the report from the Workshop 2004 in Washington, DC. Use of uridine in any form is experimental at this time. Results from the ongoing clinical studies should provide further information.
Uridine is a biological compound essential for the synthesis of DNA and RNA, and numerous other factors essential for cell metabolism. Uridine is synthesized by the power plant of the human cell known as the mitochondria. Uridine is a particular "nucleoside" which is used by our body to produce DNA. Uridine is also required for many other metabolic pathways - for example uridine is needed to produce glycogen. Uridine is a natural substance in our body. Humans are normally able to produce uridine, but the ability to do so requires intact mitochondria. Uridine is one of four nucleosides used in genetic coding for RNA, and its complement is the nucleoside adenosine.
Uridine is available commercially as a dietary supplement (NucleomaxX (http://www.nucleomaxx.com). Dr. Walker and colleagues conducted in vitro studies on hepatocytes exposed to NRTIs and found that severe depletion of mtDNA, reduction of cell proliferation, and increases in lactate levels and steatosis occurred with exposure to stavudine and zalcitabine (but not didanosine) and was largely reversed in the presence of uridine. In these studies, uridine was delivered via a nutritional supplement derived from sugar cane called Mitocnol. This agent does not appear to interfere with the efficacy of NRTIs in vitro. Other current studies are evaluating the utility of Nucleomax XTM, a new dietary supplement that contains Mitocnol, in abrogating mitochondrial toxicity associated with NRTI therapy in both preventive and therapeutic settings.
In a poster at the Workshop Walker reported on "Uridine Pharmacokinetics of Mitocnol, a Sugar Cane Extract". In vitro data and limited in vitro data indicate that the supplementation of uridine may be beneficial in preventing and treating the mitochondrial toxicity of pyrimidine NRTI by reversing mtDNA depletion. NecleomaxX is a food supplement of potential use in treatment of mitochondrial toxicity as it contains Mitocnol, a sugar cane extract with a high percentage of nucleosides. However, the exact effects of Mitocnol consumption on the serum levels of uridine in human are not known. Healthy, fasting, adult humans (4 men, 4 females) consumed 36 g of NucleomaxX by drinking 200 mg of orange juice in which the extract was dissolved. Baseline serum levels of uridine were measured by HPLC before drinking NucleomaxX and during the following 24 hours. Mean uridine serum levels at baseline were 5.6 μM (males 5.8 μM, females 5.4 μM). after NucleomaxX consumption, uridine serum levels rose sharply and peaked after 1.3 hrs. The mean maximal uridine serum concentration (Cmax) was 152.0 μM. The Cmax range was 116.0-212.0 μM. the mean Cmax in females was slightly but not significantly higher (165.4 μM) compared with males (138.6 μM), possibly due to the lower body weight and body surface area of the former (mean body weight and body surface of females: 62.3 kg and 1.71 m2) compared with the latter (males: 77.5 kg and 1.96 m2, respectively). Uridine was eliminated from the serum with an initial half-life of 2 hrs and a terminal half-life of 11.1 hrs. After 8 and 24 hrs, mean uridine serum levels were 19.3 μM and 7.5 μM, respectively. The mean AUC calculated with the linear trapezoidal rule between the time points in the time data range was 736 μMh and was identical between sexes. Adverse events were not observed. Walker concluded that Mitocnol effectively increases uridine serum levels in humans.
"Uridine Abrogates the Adverse Effects of d4T and DDC on Adipose Cell Functions"
Walker (Medizinische Universitatsklink, Frieburg, Germany) presented results from a study assessing whether uridine can prevent the adverse effect of d4T and ddC on adipocyte (fat cells) function in vitro. Walker concluded that uridine supplementation protects adipocytes in culture (fat cells in the test tube) from the adverse effects of d4T and ddC on lipid accumulation, cell survival and mitochondrial functions. The beneficial effect of uridine is seen whatever the parameter tested, suggesting that the toxic effects of these NRTIs could be linked to depletion of uridine or its metabolites inside the cells. Elevating the intracellular uridine level could allow competition of uridine metabolites with NRTIs at polymerase-gamma and thus prevent the decreased mtDNA synthesis. Uridine is an interesting candidate in the prevention of the NRTI-induced lipoatrophy in patients.
Walker provided this background in his talk. Mitochondrial DNA (mtDNA) depletion represents an attractive hypothesis to explain the subcutaneous fat wasting in HIV-infected patients (lipoatrophy) because d4T and AZT are linked to peripheral fat loss in clinical trials; d4T and AZT are associated with mtDNA depletionand increased apoptosis (cell death) in peripheral fat. Thymidine analogs such as d4T, ddC, and AZT decrease adipocyte survival, lipid content, and fat metabolism, and mitochondrial activity in cultured adipocytes.
In vitro and in vivo data suggest that uridine may safely prevent and reverse the mitochondrial toxicity of thymidine analogue reverse transcriptase inhibitors in different cell and organ systems.
3T3-F442A preadipocytes were exposed to d4T (10 μM) or ddC (0.2 μM) in the absence or presence of uridine (200 μM) for 21 days before, and 7 days after induction of differentiation. Lipid accumulation (oil red staining), apoptosis ((flow cytometry), mitochondrial DNA levels and mitochondrial membrane potential (JC-1) were evaluated at day 7 of differentiation.
Prolonged treatment with d4T or ddC markedly altered adipose cell morphology. Adipocytes were enlarged and contained lipid droplets of reduced size and number. D4T & ddC significantly decreased lipid accumulation (by 36% & 20%, respectively) and increased apoptosis (by 5.6 and 2.2 fold, respectively). D4t and ddC markedly decreased adipocyte mtDNA to residual amounts of 36% and 45%, respectively. Both NRTIs also induced mitochondrial depolarization, as shown by the 40-45% decrease of the JC-1 agregate/monomoer ratio.
Uridine had no intrinsic effect, but did away with the adverse effects of both NRTIs on adipocyte morphology and lipid staining. Uridine normalized apoptotic indices (1.1 fold for both NRTIs). Uridine prevented mtDNA-depletion by d4T and ddC (mtDNA-levels 101% & 78% of control adipocytes, respectively), and normalized mitochondrial respiration. D4T & ddC depleted mtDNA in cell culture in Walker's study but when uridine was added mtDNA was improved equal to that in controls.
URIDINE REPORT FROM THE PARIS 2003 LIPODYSTROPHY WORKSHOP
Uridine Improves Mitochondrial Toxicity In Vitro (in the laboratory)
Written by Jules Levin
The purpose of the uridine study was to evaluate whether it may be suitable to prevent and treat nucleoside reverse transcriptase inhibitor related mitochondrial toxicity. This was tested in vitro, in the laboratory. Human HepG2-hepatocytes were exposed to NRTIs with or without uridine for 25 days. DDC was used and produced a severe mtDNA depletion in this experiment. After inserting uridine into the cell culture mitochondrial toxicity was significantly improved and damage was reversed. These effects were dose-dependent and maximal at 200 uM of uridine. Uridine also rapidly and fully restored cell function despite continued ddC exposure, when added to cells displaying severe mitochondrial dysfunction. Similar results were found in HepG2 cells exposed to 36 uM of d4T, but not to 11.8 uM of ddI. Uridine also fully improved the increase in lactate and all the cell toxicity of AZT (7 uM) plus 3TC (8 uM) to HepG2 cells. Walker went on to explain that uridine appears to be safe. Uridine 150 mg/day is recommended for lifetime in patients with hereditary orotic aciduria. Oral dosing was limited by diarrhea at excessive doses when given through peripheral veinÑphlebitis. Walker reported that Mitocnol (also called NucleomaxX, an extract from sugar cane, is offered as a nutritional supplement and can be taken orally. Walker reported on one patient he treated with this product. The patient is a 50 yr-old man taking HAART that included d4T and had hepatitis, steatosis (fatty liver), CK elevation, and elevated lactate. The patient took NucleomaxX 3 sachets/day for 4 days, and Walker reported resolution of his symptoms. Walker also reported that NucleomaxX achieves high uridine serum levels. HOWEVER, this product has yet to be tested in humans with HIV for safety and effectiveness, or for potential drug interactions. Walker is planning additional study to begin next week and if efficacy is shown it's expected the product will be tested in patients with lipoatrophy.
URIDINE REPORT from the Paris Lipodystrophy Workshop
Written by Cecilia Shikuma, MD, University of Hawaii
The lack of effective treatment modalities for NRTI-induced toxicity has been a very frustrating issue in the HIV metabolic field. While the use of PPARg agonists has shown some promise, as reported already from this conference by Jules Levin, Ulrich Walker from University of Freiburg, Germany [Abs 19] presented very preliminary data on the possible efficacy of uridine to prevent and treat NRTI-mediated mtDNA depletion. Uridine is a compound occasionally utilized in patients with congenital mitochondrial diseases and is the standard treatment for hereditary orotic aciduria. In an in vitro model of human HepG1-hepatocytes observed for 25 days, ddC induces a severe depletion of mtDNA to 8% of control, a drop in respiratory chain subunit COX II to 8% of control, a severe intracellular steatosis and an increase in lactate to 350% of control. The addition of uridine with ddC resulted in repletion of mtDNA to 65% of control levels as well as full normalization of cell proliferation, lactate levels and intracellular lipid levels. Even more impressive was the fact that uridine could be used in a "simulated" in vitro "treatment" mode to substantially ameliorate such evidence of mitochondrial toxicity when it was added following 15 days of ddC exposure. Similar results were obtained when ZDV or d4T was used instead of ddC. Uridine did not alter the IC50 or IC90 of currently licensed NRTIs in HIV resistance assays. A uridine compound extracted from sugar cane named Mitocnol (NucleomaxX¨) is available for clinical use. The chief known side effect of this medication is diarrhea when given orally. Dr. Walker reported use of this agent in a single 50 yr old subject on d4T containing ARV regimen who exhibited evidence of hepatitis, steatosis, CK elevation with a lactate level of 4.2. Treatment for 4 days with Mitocnol resulted in a substantial improvement in these parameters. A phase I clinical trial is planned. While the in vitro data presented by Dr. Walker requires independent confirmation and treatment in one subject may have little value in determining efficacy, further research on the potential use of uridine will certainly be watched with interest.
Beneficial effects of oral uridine in mitochondrial toxicity
Correspondence in AIDS: Volume 18(7) 30 April 2004 pp 1085-1086
Walker, Ulrich Aa; Langmann, Peterb; Miehle, Nikolausa; Zilly, Michaelb; Klinker, Hartwigb; Petschner, Franziskaa
aMedizinische Universitätsklinik, Department of Rheumatology and Clinical Immunology, Freiburg, Germany; and bUniversity of Wuerzburg, Department of Infectious Diseases, Wuerzburg, Germany.Received: 19 August 2003; revised: 11 September 2003; accepted: 16 September 2003.
Note from Jules Levin: Data on this treatment was reported at the Lipodystrophy Workshop in Paris in 2003. It's my understanding that clinical studies are underway.
Some long-term side-effects of antiretroviral therapy are now attributed to the mitochondrial toxicity of nucleoside analogue reverse transcriptase inhibitors (NRTI) and specifically their ability to deplete mitochondrial DNA. Using the human hepatoma (HepG2) cell line, we have discovered that the nucleoside uridine prevents and treats mtDNA depletion by pyrimidine NRTI (such as zalcitabine or stavudine). In this hepatocyte model, uridine increased mtDNA levels and fully abrogated consequent hepatocyte death, elevated lactate production and intracellular steatosis. Several investigators have also found beneficial effects of uridine in zidovudine-related haematopoietic toxicity, both in vitro and in animals. Depending on the system studied, uridine was effective at concentrations of 50-200 M. Various steps of mitochondrial nucleoside utilization may be involved in the protective effect, but the competition of uridine or its metabolites with NRTI at gamma-polymerase is the most plausible explanation. Pharmacokinetic studies of uridine in humans suggest that high uridine serum concentrations can be achieved by oral or intravenous dosing. Oral dosing is limited by a mild osmotic diarrhoea at 10-12 g/m2 uridine. Uridine supplementation was also safely used long term in numerous patients suffering from hereditary orotic aciduria, an inborn error of pyrimidine de-novo synthesis. Investigations performed in vitro and in mice indicated that high concentrations of uridine do not interfere with the antiretroviral activity of the NRTI.
Taken together, these data suggest that strategies aimed at increasing uridine may be promising in the treatment of some symptomatic or life-threatening mitochondrial toxicities in HIV-infected patients.NucleomaxX is a food supplement consisting of mitocnol, an extract from sugar canes with a high content (17%) of nucleosides (http://www.nucleomaxX . com). Drinking the contents of a single sachet (36 g) of NucleomaxX increases the physiological serum concentration of uridine in humans from approximately 5 mM to more than 100 mM. We have used NucleomaxX first in an HIV patient with mitochondrial steatohepatitis and symptomatic elevation of creatine kinase under long-term antiretroviral treatment with stavudine.
A 54-year-old caucasian man (168 cm, 75 kg) was started on antiretroviral medication 4 years ago, when he was diagnosed with HIV-1/AIDS as a result of Pneumocystis carinii pneumonia, pulmonary tuberculosis and oesophageal candidiasis. When he first presented to our institution 2 years ago, his initial CD4 cell count of 25 cells/ml had risen to 682 cells/ml and his previous stavudine-containing antiretroviral regimen had been switched to lamivudine 150 mg twice a day, stavudine 40 mg twice a day, abacavir 300 mg twice a day and efavirenz 600 mg a day. We continued this regimen because side-effects were not clinically apparent initially and because HIV was undetectable in the blood. After several months, he developed myalgias, and a continuous increase in creatine kinase, lactate, and transaminases. Tests for hepatotropic viruses, alpha-1 antitrypsin deficiency and haemochromatosis were negative, and there was no indication of alcohol abuse. There was no indication of cholestasis, but abdominal ultrasound revealed signs of massive liver steatosis. The patient was then started on NucleomaxX for suspected stavudine-related mitochondrial toxicity (three sachets a day for 4 days). After 2 weeks, at his next visit, liver and muscle enzymes as well as the myalgias had improved rapidly, despite unchanged medication. Lactate had normalized after 7 weeks. Stavudine was then switched to tenofovir. There were no subsequent clinical or laboratory abnormalities, HIV replication remained below the limit of detection (< 50 copies/ml blood) and ultrasound showed a substantial improvement of steatotic signs.
References
1. Walker UA, Koch E, Venhoff N, Klinker H, Langman P, Zilly M, et al. Uridine prevents and treats mtDNA-depletion by NRTI- pyrimidine analogues and fully restores mitochondrial function. Antiviral Ther 2003; 8:L17.
2. Walker UA, Venhoff N, Koch E, Olschweski M, Schneider J, Setzer B. Uridine abrogates mitochondrial toxicity related to nucleoside analogue reverse transcriptase inhibitors in HepG2 cells. Antiviral Ther 2003; 8:463-470.
3. Sommadossi JP, Carlisle R, Schinazi RF, Zhou Z. Uridine reverses the toxicity of 3'-azido-3'-deoxythymidine in normal human granulocyte-macrophage progenitor cells in vitro without impairment of antiretroviral activity. Antimicrob Agents Chemother 1988; 32:997-1001.
4. Calabresi P, Falcone A, St Clair MH, Wiemann MC, Chu SH, Darnowski JW. Benzylacyclouridine reverses azidothymidine-induced marrow suppression without impairment of anti-human immunodeficiency virus activity. Blood 1990; 76:2210-2215.
5. Leyva A, van Groeningen CJ, Kraal I, Gall H, Peters GJ, Lankelma J, Pinedo HM. Phase I and pharmacokinetic studies of high-dose uridine intended for rescue from 5-fluorouracil toxicity. Cancer Res 1984; 44:5928-5933.
6. van Groeningen CJ, Leyva A, Kraal I, Peters GJ, Pinedo HM. Clinical and pharmacokinetic studies of prolonged administration of high-dose uridine intended for rescue from 5-FU toxicity. Cancer Treat Rep 1986; 70:745-750.
7. van Groeningen CJ, Peters GJ, Nadal JC, Laurensse E, Pinedo HM. Clinical and pharmacologic study of orally administered uridine. J Natl Cancer Inst 1991; 83:437-441.
8. Girot R, Hamet M, Perignon JL, Guesnu M, Fox RM, Cartier P, et al. Cellular immune deficiency in two siblings with hereditary orotic aciduria. N Engl J Med 1983; 308:700-704.
References from www.mitocnol.com
Mitocnol Research
(1) DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med 2003; 348:2656-2668
(2) Brinkman K, ter Hofstede HJ, Burger DM, Smeitink JA, Koopmans PP. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway. AIDS 1998; 12:1735-1744.
(3) Brinkman K, Smeitink JA, Romijn JA, Reiss P. Mitochondrial toxicity induced by nucleoside-analogue reverse-transcriptase inhibitors is a key factor in the pathogenesis of antiretroviral-therapy-related lipodystrophy. Lancet 1999; 354:1112-1115.
(4) Kakuda TN. Pharmacology of nucleoside and nucleotide reverse transcriptase inhibitor-induced mitochondrial toxicity. Clin Ther 2000; 22:685-708.
(5) Walker UA, Koch E, Venhoff N, Klinker H, Langman P, Zilly M et al. Uridine prevents and treats mtDNA-depletion by NRTI-pyrimidine analogues and fully restores mitochondrial function. Antivir Ther 2003; 5th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV, Paris.
(6) Venhoff N, Setzer B, Lebrecht D, Walker UA. Dietary supplements in the treatment of NRTI-related mitochondrial toxicity. AIDS 2002; 16:800-802.
(7) Walker UA, Byrne E. The therapy of respiratory chain encephalomyopathy: a critical review of the past and current perspective. Acta Neurol Scand 1995; 92:273-280.
(8) Sommadossi JP, Carlisle R, Schinazi RF, Zhou Z. Uridine reverses the toxicity of 3'-azido-3'-deoxythymidine in normal human granulocyte-macrophage progenitor cells in vitro without impairment of antiretroviral activity. Antimicrobial Agents & Chemotherapy 1988; 32(7):997-1001.
(9) Cui L, Locatelli L, Xie MY, Sommadossi JP. Effect of nucleoside analogs on neurite regeneration and mitochondrial DNA synthesis in PC-12 cells. J Pharmacol Exp Ther 1997; 280:1228-1234.
(10) Herrmann ML, Schleyerbach R, Kirschbaum BJ. Leflunomide: an immunomodulatory drug for the treatment of rheumatoid arthritis and other autoimmune diseases. Immunopharmacology 2000; 47:273-289.
(11) Setzer B, Schlesier M, Thomas AK, Walker UA. Immunosuppressive effect of mtDNA-depletion by d-drugs in healthy human-lymphocytes. Antivir Ther 2003; 5th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV, Paris.
(12) Eva Koch, Josef Schneider, Roland Weis, Björn Penning and Ulrich A. Walker. Uridine excess does not interfere with the antiretroviral efficacy of nucleoside analogue reverse transcriptase inhibitors. Antiviral Therapy 2003; 8: 485-487.
(13) Ulrich A. Walker, Nils Venhoff, Eva koch, Manfred Olschewski, Josef Schneider and Bernard Setzer. Uridine abrogates mitochondrial toxicity related to nucleoside analogue reverse transcriptase inhibitors in HepG2 cells. Antiviral Therapy 2003; 8: 87-94.
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