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Ulrich A. Walker and Grace A. McComsey

Two years after the introduction of protease inhibitors into the armamentarium of antiviral therapy, reports of HIV-infected individuals experiencing clinically relevant changes in body metabolism began to surface. These "metabolic" symptoms were initially summarized under the term "lipodystrophy" (Carr 1998). Today, ten years after the introduction of highly active antiretroviral therapy (HAART), this lipodystrophy syndrome is increasingly understood as the result of overlapping, but distinct effects of the different drug components within the HAART antiretroviral cocktail. The main pathogenetic mechanism through which nucleoside analogs are thought to contribute to the metabolic changes and organ toxicities is mitochondrial toxicity (Brinkman 1999).

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Pathogenesis of mitochondrial toxicity NRTIs are prodrugs (Kakuda 2000) because they require activation in the cell through phosphorylation before they are able to inhibit their target, e.g. HIV reverse transcriptase. In addition to impairing the HIV replication machinery, the NRTI-triphosphates also inhibit a human polymerase called "polymerase-gamma", which is responsible for the replication of mitochondrial DNA (mtDNA). Thus, the inhibition of polymerase-gamma by NRTIs leads to a decline (depletion) in mtDNA, a small circular molecule normally present in multiple copies in each mitochondrion and in hundreds of copies in most human cells (Lewis 2003). The only biological task of mtDNA is to encode for enzyme subunits of the respiratory chain, which is located in the inner mitochondrial membrane. Therefore, by causing mtDNA-depletion, NRTIs also lead to a defect in respiratory chain function. An intact respiratory chain is the prerequisite for numerous metabolic pathways. The main task of the respiratory chain is to oxidatively synthesize ATP. In addition, the respiratory chain consumes NADH and FADH as end products of fatty acid oxidation. This fact explains the micro- or macrovesicular accumulation of intracellular triglycerides, which often accompanies mitochondrial toxicity. Last but not least, a normal respiratory function is also essential for the synthesis of DNA, because the de novo synthesis of pyrimidine nucleosides depends on an enzyme located in the inner mitochondrial membrane. This enzyme is called dihydroorotate dehydrogenase (DHODH) (Löffler 1997). The clinical implications of this fact are detailed below. The onset of mitochondrial toxicity follows certain principles (Walker 2002a): 1. Mitochondrial toxicity is concentration dependent with high NRTI-concentrations causing a more pronounced mtDNA-depletion. The clinical dosing of some nucleoside analogs is close to the limit of tolerance with respect to mitochondrial toxicity. 2. The onset of mitochondrial toxicity requires time. Changes in mitochondrial metabolism are observed only if the amount of mtDNA-depletion exceeds a certain threshold, an effect observed solely with prolonged NRTI-exposure. As a consequence of this effect, the onset of mitochondrial toxicity is typically not observed clinically in the first few months of HAART. Furthermore, long-term NRTI exposure may also lead to mitochondrial effects despite relatively low NRTI concentrations. 3. There are significant differences in the relative potencies of nucleoside and nucleotide analogs in their ability to interact with polymerase-gamma. The hierarchy of polymerase-gamma inhibition for the active NRTI metabolites has been determined as follows: ddC (HIVIDTM) > ddI (VidexTM) > d4T (ZeritTM) > 3TC (EpivirTM) = ABC (Ziagen™) = TDF (Viread™) = FTC (Emtriva™). 4. AZT may be peculiar because its active triphosphate is only a weak inhibitor of polymerase-gamma. However, another mechanism can explain how AZT could cause mtDNA-depletion independent from polymerase-gamma inhibition. AZT is an inhibitor of mitochondrial thymidine kinase type 2 (TK2), and, as such, interferes with the synthesis of natural pyrimidine nucleotides, thus potentially impairing the formation of mtDNA (McKee 2004, Saada 2001). AZT may also be metabolized to d4T, at least within some cells in our body (Becher 2003, Bonora 2004). 5. Mitochondrial toxicity is tissue specific. Tissue specificity is explained by the fact that the uptake of the NRTIs into cells and their mitochondria, as well as activation by phosphorylation may be different among individual cell types. 6. There may be additive or synergistic mitochondrial toxicities if two or more NRTIs are used in combination. 7. Mitochondrial transcription may also be impaired without mtDNA-alterations (Mallon 2005, Galluzzi 2005). The mechanism and the clinical significance of this observation are however not yet understood. Clinical manifestations MtDNA-depletion may manifest clinically in one or several main target tissues (Fig. 1). In the liver mitochondrial toxicity is associated with increased lipid deposits, resulting in micro or macrovesicular steatosis. Steatosis may be accompanied by elevated liver transaminases. Such steatohepatitis may be observed with even one NRTI such as DDI and progress to liver failure and lactic acidosis, a potentially fatal, but fortunately rare complication (Lambert 1990). Hepatotoxicity is now predominantly observed under treatment with dideoxynucleosides, i.e. with ddI, d4T, and ddC, but also with AZT. The onset of hepatic mtDNA-depletion is dependent on the time of NRTI exposure (Walker 2004a). On electron microscopy, morphologically abnormal mitochondria are observed. Figure 1: Organ manifestations of mitochondrial toxicity. The question marks signify manifestations, which are still under debate. A typical complication of mitochondrial toxicity is an elevation in serum lactate. Such hyperlactatemia was more frequently described with prolonged d4T treatment (Saint-Marc 1999, Carr 2000), especially when combined with ddI. The toxicity of ddI is also increased through the interactions with ribavirin and hydroxyurea. The significance of asymptomatic hyperlactatemia is unclear. When elevated lactate levels are associated with symptoms, these are often non-specific (nausea, right upper quadrant abdominal tenderness, myalgias). In the majority of cases, the serum bicarbonate levels and the anion gap (Na+ - [HCO3- + Cl-]) are normal, although liver transaminases are mildly elevated in the majority of cases. Therefore, the diagnosis relies on the logistically more cumbersome direct determination of serum lactate. In order to avoid artifacts, venous blood must be drawn without the use of a tourniquet from resting patients. The blood needs to be collected in fluoride tubes and transported to the laboratory on ice for immediate analysis. Non-mitochondrial causes must also be considered in the differential diagnosis of lactic acidosis (Table 1) and underlying organ toxicities should be looked for. Table 1. Causes of hyperlactatemia/ lactic acidosis Type A lactic acidosis Type B lactic acidosis (Tissue hypoxia) Shock Carbon monoxide poisoning Heart failure (Other mechanisms) Thiamine deficiency Alkalosis (pH>7.6) Epilepsy Adrenalin (iatrogenic, endogenous) Liver failure Neoplasm (lymphoma, solid tumors) Intoxication (nitroprusside, methanol, methylene glycol, salicylates) Fructose Rare enzyme deficiencies mtDNA mutations mtDNA depletion A mitochondrial myopathy in antiretrovirally treated HIV patients was first described with high dose AZT therapy (Arnaudo 1991). Skeletal muscle weakness may manifest under dynamic or static exercise. The serum CK is often normal or only minimally elevated. Muscle histology helps to distinguish this form of NRTI toxicity from HIV myopathy, which may also occur simultaneously. On histochemical examination, the muscle fibers of the former are frequently negative for cytochrome c-oxidase and carry ultrastructurally abnormal mitochondria, whereas those of the latter are typically infiltrated by CD8+ T-lymphocytes. Exercise testing may detect a low lactate threshold and a reduced lactate clearance, but in clinical practice these changes are difficult to distinguish from lack of aerobic exercise (detraining). Prolonged treatment with D-drugs may also frequently lead to a predominantly symmetrical, sensory and distal polyneuropathy of the lower extremities (Simpson 1995, Moyle 1998). An elevated serum lactate level may help to distinguish this axonal neuropathy from its HIV-associated phenocopy, although in most cases the lactate level is normal. The differential diagnosis may also take into account the fact that the mitochondrial polyneuropathy mostly occurs weeks or months after initiation of D-drugs. In contrast, the HIV-associated polyneuropathy generally does not worsen and may indeed improve with prolonged antiretroviral treatment. In its more narrow sense, the term "lipodystrophy" denotes a change in the distribution of body fat under prolonged HAART exposure. Some subjects affected with lipodystrophy may experience abnormal fat accumulation in certain body areas (most commonly abdomen or dorsocervical region), whereas others may develop fat wasting (Bichat's fat pad in the cheeks, temporal fat, or subcutaneous fat of the extremities). Both fat accumulation and fat loss may at times occur simultaneously in the same individuals. Fat wasting (also called lipoatrophy) is partially reversible and generally observed not earlier than one year after the initiation of HAART. In the affected subcutaneous tissue, ultrastructural abnormalities of mitochondria and reduced mtDNA levels have been identified, in particular in subjects treated with d4T (Walker 2002b). In vitro and in vivo analyses of fat cells have also demonstrated diminished intracellular lipids, reduced expression of adipogenic transcription factors (PPAR-gamma and SREBP-1), and increased apoptotic indices. NRTI treatment may also impair some endocrine functions of adipocytes. For example, they may impair the secretion of adiponectin and through this mechanism may promote insulin resistance. d4T has been identified as a particular risk factor, but other NRTIs such as AZT may also contribute. When d4T is replaced by another NRTI, mtDNA-levels and apoptotic indices improve along with an objectively measurable, albeit small increase of subcutaneous adipose tissue (McComsey 2004). In contrast, switching away from PIs did not ameliorate lipoatrophy and adipocyte apoptosis. Taken together, the available data point towards a predominant effect of NRTI-related mitochondrial toxicity in the pathogenesis of lipoatrophy. Some studies have suggested an effect of NRTIs on the mtDNA levels in blood (Coté 2003, Miro 2003). The functional consequence of such mitochondrial toxicity on lymphocytes is still unknown. In this context, it is important to note that a delayed loss of CD4+ and CD8+ T-lymphocytes was observed, when ddI plasma levels were increased by comedication with TDF, or by low body weight (Negredo 2004). Exposure of mitotically stimulated T-lymphocytes to slightly supratherapeutic concentrations of ddI also demonstrated a substantial mtDNA-depletion with a subsequent late onset decline of lymphocyte proliferation and increased apoptosis (Setzer 2005). These data suggest that the mitochondrial toxicity of NRTIs on lymphocytes is responsible for the late onset decline of lymphocytes observed with ddI and has immunosuppressive properties. Asymptomatic elevations in serum lipase are not uncommon under HAART, but of no value in predicting the onset of pancreatitis (Maxson 1992). The overall frequency of pancreatitis has been calculated as 0.8 cases/ 100 years of NRTI-containing HAART. Clinical pancreatitis is associated with the use of ddI in particular. ddI reexposure may trigger a relapse and should be avoided. A mitochondrial mechanism to explain the onset of pancreatitis has been hypothesized but remains unproven. Prolonged treatment with didexoynucleosides is also associated with hyperuricemia (Walker 2006a). The mechanism may be two-fold. Mitochondrial dysfunction may increase the formation of lactate, which competes with urate for tubular secretion in the kidney. Respiratory chain failure also causes ATP depletion, which is known to increase urate production in the purine nucleotide cycle. The existence of mitochondrial damage to the kidney is controversial. Supratherapeutic doses of TDF (VireadTM) induced a Fanconi syndrome with tubular phosphate loss and consecutive osteomalacia in animals (Tenofovir review team 2001). TDF is a nucleotide analogue and taken up into the renal tubules by means of a special anion transporter. Excessive intratubular drug concentrations may impair mtDNA replication, despite the fact that TDF is only a weak inhibitor of polymerase-gamma. Decreased mtDNA levels have recently been found in renal biopsies from patients exposed to TDF plus ddI, a NRTI combination that for several reasons is no longer recommended (Côté 2006). It should be noted that neither the trials leading to the approval of TDF, nor the subsequent field data were able to prove the mitochondrial nephrotoxicity of TDF. Most trials only measured creatinine clearance and serum phosphate (Izzedine 2005) although a compromise in renal function is not expected in Fanconi's syndrome and increased renal phosphate loss may be masked by preserved by homeostatic phosphate mobilization from bone. More sensitive methods have recently revealed a diminished renal phosphate reabsorption and an elevated alkaline phosphatase in patients treated with TDF (Kinai 2005). Cases of phosphate diabetes were also reported under treatment with other NRTIs. ZDV is also used to reduce the risk of HIV vertical transmission and in this setting was associated with low mtDNA levels in the placenta and in the peripheral cord blood of neonates (Shiramizu 2003, Divi 2005). ZDV also causes a transient anemia in the newborn, as well as neutropenia, thrombopenia and lymphopenia, which may persist for months (Venhoff 2006). A French cohort found an increased frequency of mitochondrial myopathies in infants perinatally exposed to NRTIs (Blanche 1999). Hyperlactatemia is not infrequently observed in the perinatal setting and may persist for several months after delivery (Noguera 2003). Long-term data are lacking and better surveillance systems should be implemented (Venhoff 2006). Monitoring and diagnosis There is currently no method to reliably predict the mitochondrial risk of an individual. The quantification of mtDNA-levels in the peripheral blood is not useful. Quantifying mtDNA within affected tissues is likely to be more sensitive; but invasive and not prospectively evaluated with regard to clinical endpoints. Once symptoms are established, histological examination of a tissue biopsy may contribute to the correct diagnosis. The following findings in tissue biopsies point towards a mitochondrial etiology: ultrastructural abnormalities of mitochondria, diminished histochemical activities of cytochrome c-oxidase, the detection of intracellular and more specifically microvesicular steatosis, and the so-called ragged-red fibers. Treatment and prophylaxis of mitochondrial toxicity Drug interactions Drug interactions may precipitate mitochondrial symptoms and must be taken into account. The mitochondrial toxicity of ddI for example is augmented through drug interactions with ribavirin, hydroxyurea and allopurinol (Ray 2004). When ddI is combined with TDF, the ddI dose must be reduced to 250 mg QD. The thymidine analog brivudine is a herpes virostatic that may sensitize for NRTI-related mitochondrial toxicity because one of its metabolites is an inhibitor of DHODH (see below). Brivudine should therefore not be combined with antiretroviral pyrimidine analogues. An impairment of mitochondrial metabolism may also result from ibuprofen, valproic acid and acetyl salicylic acid as these substances impair the mitochondrial utilization of fatty acids. Acetyl salicylic acid may damage mitochondria and such damage to liver organelles may result in Reye's syndrome. Valproic acid may trigger a life threatening lactic acidosis. Amiodarone and tamoxifen also inhibit the mitochondrial synthesis of ATP. Acetaminophen and other drugs impair the antioxidative defense (glutathione) of mitochondria, allowing for their free radical mediated damage. Aminoglycoside antibiotics and chloramphenicol not only inhibit the protein synthesis of bacteria, but under certain circumstances also impair the peptide transcription of mitochondria as bacteria-like endosymbionts. Adefovir and cidofovir are also inhibitors of polymerase-gamma. Alcohol is also a mitochondrial toxin. The most important clinical intervention is the discontinuation of the NRTI(s) responsible for mitochondrial toxicity. Randomized studies have demonstrated that switching d4T to a less toxic alternative leads to a slight and slowly progressive improvement in lipoatrophy (McComsey 2004, Martin 2004, Moyle 2006). Switching away from PIs to NNRTIs however was not associated with an improvement of lipoatrophy. These findings stress the crucial role of mitochondrial toxicity in the pathogenesis of fat wasting. Figure 2: Mechanism of Mitocnol (NucleomaxXTM) in the prevention and treatment of mitochondrial toxicity. Uridine The so far only therapy of mitochondrial toxicity under unchanged NRTI-treatment consists of the supplementation of uridine or its precursors. As outlined above, any respiratory chain impairment also results in the inhibition of DHODH, an essential enzyme for the synthesis of uridine and its derived pyrimidines (Fig 2). This decrease in intracellular pyrimidine pools leads to a relative excess of the exogenous pyrimidine nucleoside analogs, with which they compete at polymerase-gamma. A vicious circle is closed and contributes to mtDNA-depletion. By supplementing uridine this vicious circle can be interrupted, resulting in increased mtDNA-levels. Indeed, uridine abolished in hepatocytes all the effects of mtDNA-depletion and normalized lactate production, cell proliferation, the rate of cell death and intracellular steatosis. (Walker 2003). In contrast, vitamin cocktails were not beneficial in this model. Uridine also normalizes the lipoatrophic phenotype in adipocytes exposed to d4T (Walker 2006b). Uridine is well tolerated by humans, even at high oral and intravenous doses (van Groeningen 1986, Kelsen 1997). A food supplement called Mitocnol was shown to have a more than 8-fold uridine bioavailability over conventional uridine (Venhoff 2005). Mitocnol was studied in a randomized placebo-controlled double-blind trial in lipoatrophic subjects under continued therapy with d4T or AZT where it has objectively improved subcutaneous fat (Sutinen 2007). In comparison with switch strategies (e.g. the replacement of stavudine and zidovudine by antivirals with a reduced potential of mitochondrial toxicity), the effect of Mitocnol on subcutaneous fat gain was more rapid and quantitatively more pronounced (Fig 3). Figure 3: Subcutaneous fat gain with Mitocnol under d4T and AZT treatment (in comparison with strategies sparing thymidine-analogue NRTI). A second trial has also suggested Mitocnol to be efficacious with regard to patient and physician assessed lipoatrophy scores (McComsey 2007). In vitro, animal and clinical data indicate, that Mitocnol also antagonizes mitochondrial steatohepatitis. (Walker 2004b, Banasch 2006, Lebrecht 2007). Animal data indicate that uridine supplementation also counteracts AZT-induced hematotoxicity and myopathy (Sommadossi 1988). Mitocnol is well tolerated and adverse events have not been observed so far. In one study, a clinically insignificant HDL-decline was suggested, while another trial showed no change in HDL cholesterol (McComsey 2007). There are no known negative interactions of uridine with the efficacy of the antiretroviral treatment (Sommadossi 1988, Koch 2003, McComsey 2007, Sutinen 2007). In Europe and North America, Mitocnol is available as a dietary supplement called NucleomaxX® and can be acquired in pharmacies and the internet (www.nucleomaxX.com). In symptomatic hyperlactatemia and in lactic acidosis, all NRTIs should be immediately discontinued (Brinkman 2000). The supplementation of vitamin cocktails has been recommended, but there are no data that demonstrate the efficacy of this intervention with respect to mtDNA-depletion (Walker 1995, Venhoff 2002). After discontinuation of NRTIs, normalization of lactate may require several weeks. More mitochondrial friendly NRTIs may then be reintroduced, but patients should be monitored closely. The proposed supportive treatment of hyperlactatemia and lactic acidosis is summarized in Table 2. Table 2. Supportive treatment of lactate elevation in HIV-infected patients (non-pregnant adults) Lactate 2-5 mmol/L + symptoms Lactate > 5 mmol/L or lactic acidosis Discontinue mitochondrial toxins Consider vitamins and NucleomaxX (36g TID on 3 consecutive days/ month) Discontinue NRTIs and all mitochondrial toxins Intensive care Maintain hemoglobin > 100 g/L Avoid vasoconstrictive agents Oxygen Correct hypoglycemia Bicarbonate controversial - 50-100 mmol if pH<7.1 Coenzyme Q10 (100 mg TID) Vitamin C (1 g TID) Thiamine (Vit. B1, 100 mg TID) Riboflavin (Vit. B2, 100 mg QD) Pyridoxine (Vit. B6, 60 mg QD) L-acetyl carnitine (1 g TID) NucleomaxX (36 g TID until lactate <5 mmol/L) References 1. Arnaudo E, Dalakas M, Shanske S, Moraes CT, DiMauro S, Schon EA. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 1991, 337:508-10. http://amedeo.com/lit.php?id=1671889 2. Banasch M, Goetze O, Knyhala K et al. 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Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine. Hepatology 2004a, 39:311-17. http://amedeo.com/lit.php?id=14767983 52. Walker UA, Langmann P, Miehle et al. Beneficial effects of oral uridine in mitochondrial toxicity. AIDS 2004b, 18:1085-6. http://amedeo.com/lit.php?id=15096820 53. Walker UA, Hofmann C, Enters M, et al. High serum urate in HIV-infected persons. The choice of the antiretroviral drug matters. AIDS 2006a, 13: 1556-8. http://amedeo.com/lit.php?id=16847413 54. Walker UA, Auclair M, Lebrecht D, Kornprobst M, Capeau J, Caron M. Uridine abrogates the adverse effects of stavudine and zalcitabine on adipose cell functions. Antivir Ther 2006b, 11: 25-34. http://amedeo.com/lit.php?id=16518957