Pathology of LipodystrophyMitochondrial Toxicity of NRTIs
A talk by Kees Brinkman from Amsterdam about mitochondrial toxicity of NRTIs presaged the rest of the meeting.[4] He advanced the hypothesis that NRTI toxicity is a common and significant phenomenon that is the result of mitochondrial toxicity. By way of background, mitochondrial DNA encodes some of the proteins involved in oxidative phosphorylation. Mitochondrial DNA is vulnerable to damage and has limited ability to repair mutations. NRTIs have been shown to affect DNA polymerase gamma, which is found in mitochondria. In vitro incubations with NRTIs over days were shown to lead to decreased mitochondrial DNA. In the assay used, the effect was greatest for ddC, less with ddI, d4T, and ZDV, and not seen with 3TC. Decreases in mitochondrial DNA are also seen with the nucleotide adefovir.Dr. Brinkman suggested that most of the clinical NRTI toxicity might be related to mitochondrial toxicity, including polyneuropathy, myopathy, steatosis, lactic acidosis, pancreatitis, pancytopenia, and proximal tubular dysfunction (the latter seen with adefovir). Variations in the toxicities associated with different NRTIs may be related to variable tissue sensitivity for NRTI damage, variable entry into cells, variable phosphorylation, and the intrinsic importance of oxidative phosphorylation in specific cell function. For example, hemolytic anemia would not be expected, since erythrocytes do not contain mitochondria.
Lactic acidosis with serious disease and poor outcome was first recognized to occur with ZDV. However, other NRTIs can produce similar problems. Clinical symptoms include nausea, vomiting, abdominal pain, and hyperventilation. Risk factors for the development of the syndrome include preceding NRTI toxicity of any sort, recent metabolic stress, possibly micronutrient deficiencies such as riboflavin, carnitine, and possibly a congenital predisposition.
The diagnosis is based upon the detection of abnormal elevations of serum lactate concentration. Random lactate levels are not recommended, and increases in anion gap are felt to be insensitive. Serum lactate following a glucose load may be the best means to detect mitochondrial toxicity. The best treatment is recognition of the syndrome and discontinuation of all NRTIs. There is suggestive evidence of clinical benefit with riboflavin therapy.
The relationship of mitochondrial toxicity to lipodystrophy is tenuous. A lipodystrophy syndrome -- multiple symmetrical lipomatosis -- was shown to involve some abnormality in mitochondrial DNA and problems in oxidative phosphorylation, specifically via cytochrome complex 4. Only a minority of HIV-infected subjects with lipodystrophy have this physique, so that the association may be coincidental or even unrelated to the pathogenesis of the lipodystrophy syndrome. Unfortunately, it appeared that many people at the meeting came away with the impression that the link between mitochondrial toxicity and lipodystrophy was strong. In fact, the hypothesis has no direct proof, much like the LRP/CRABP hypothesis,[5] and should be seen for what it is: a specific construct whose validity can be tested in prospective studies. At present, the mitochondrial hypothesis should not be a consideration in treatment decision-making.
However, those of us in the business of designing and performing clinical investigations must reconsider our current and planned studies if possible differences in outcomes between NRTIs are to be distinguished.
At the recent Retroviruses conference (Chicago, Jan. 31 - Feb. 4), a research team from Glaxo Wellcome presented two posters proposing possible mechanisms for the lipid (fat) abnormalities which appear to be associated with use of protease inhibitors in some patients--together with supporting biochemical data from laboratory studies.1,2 Other mechanisms have been proposed, but without such laboratory data. Unfortunately the posters had limited impact at the conference, because this research is based on lipid biochemistry which is unfamiliar to most AIDS experts. We have met with the researchers and received additional documentation to help us explain this work to a larger audience.Part of the difficulty in research on lipodystrophy is that pharmaceutical companies have disincentives to highlight problems with their products. Glaxo has different incentives, because it has not marketed a protease inhibitor yet--and the lipid research suggest that its protease inhibitor amprenavir (Agenerase(tm), which was originally developed by Vertex Pharmaceuticals Inc. and formerly known as VX478 or 141W94), may not have the same problem. Also, according to Glaxo, there has been almost no lipodystrophy in its expanded-access program which so far has given the drug to over 1500 patients. But no one knows if these laboratory results will predict what happens in people; and the expanded-access program has not given amprenavir to enough people for long enough to know with confidence that they will not have as much lipodystrophy as those treated with other drugs, since this problem is most likely to occur after long-term use. While these results are interesting, only time will tell whether this drug will be associated with less lipodystrophy than other HIV protease inhibitors in human use.
Mechanisms
No one knows for sure what is causing symptoms such as "Crix belly" or "buffalo hump" and associated loss of fat in the face, arms, and legs--and metabolic complications which are often associated, including high triglycerides and cholesterol, and development of insulin resistance or even diabetes in some patients. Most experts today believe that protease inhibitors are probably involved, even though there were a few such cases before these drugs were used.Many researchers believe that the fundamental problem in this syndrome is the loss of subcutaneous fat, rather than its abnormal accumulation. The loss of equilibrium between fat deposited in the body and lipids in the bloodstream may lead to very high blood levels of LDL cholesterol (the "bad cholesterol") and triglycerides. For unknown reasons, this excessive fat in the bloodstream can be deposited in certain parts of the body, causing abnormal fatty growth there--and also increasing the long-term risk of cardiovascular disease. In addition, the loss of fat due to certain illnesses is associated with a predisposition to insulin resistance--the relative ineffectiveness of insulin in the body, which can lead to diabetes if it becomes severe enough.
The Glaxo Wellcome researchers who did these studies had much previous experience in measuring lipid changes, through studies of other conditions including diabetes and obesity. They applied tests they were already familiar with to look for effects of HIV protease inhibitors on fat cells in the laboratory. Their work suggests that not all lipodystrophy is the same, but that there are at least two distinct mechanisms involved, depending on which protease inhibitor is used. (In both cases the tests were done with animal cells--a reminder that this work is still a theory of lipodystrophy, not a proven cause.)
One Possible Mechanism:
Preventing Development of Fat Cells
In one experiment,1 fat cells were grown in the laboratory, with or without HIV protease inhibitors. In this test, saquinavir, ritonavir, and nelfinavir greatly reduced the development of fat cells from stem cells; however, neither indinavir nor amprenavir had much effect. Saquinavir, ritonavir, and nelfinavir also increased the metabolic destruction of fat in existing fat cells.
Another Mechanism:
Increasing Retinoid Toxicity
Retinoids are compounds related to vitamin A; some of them are found naturally in the body, where they have many different effects. Too much of certain retinoids causes toxic effects due to abnormal biochemical signaling in the body, and the toxicity of excessive vitamin A can resemble some of those sometimes seen in the lipodystrophy syndrome.The same animal cells used above were also tested in this experiment.2 But here, instead of measuring the fat cells produced in a laboratory culture, the researchers used a certain gene in these cells, which is sensitive to retinoid signaling and also produces a product which is easy to measure in the laboratory. The protease inhibitors alone did not affect the activity of the gene.
When protease inhibitors were combined with retinoids the results were complex, depending on the protease inhibitor and the retinoid. But perhaps the most important single result is that indinavir (alone among all the protease inhibitors in human use) clearly stimulated signaling by all-trans retinoic acid (ATRA). ATRA is a retinoid produced naturally in the body from vitamin A. While it is too early to know that these laboratory studies apply to humans, the researchers suggest that indinavir may cause some lipodystrophy problems by changing retinoid signaling--in effect, causing retinoid toxicity not by increasing the amount of ATRA present, but by increasing the body's sensitivity to it.
If this theory is correct (and no one knows yet), it would suggest that vitamin A supplementation might be harmful if one is taking indinavir, by making some of the lipodystrophy problems more likely to occur. Possibly this theory could be tested by looking for correlations between lipodystrophy and vitamin A intake in clinical databases--if there are databases which have enough patients taking indinavir, keep consistent records on lipodystrophy, and record nutritional intake.
Cases of lipodystrophy have been reported among people taking any of the currently licensed protease inhibitors. In some studies the development of the signs of lipodystrophy during protease inhibitor therapy was as high as 64%.
The results of an Australian trial of combination therapy with ritonavir and saquinavir showed, 30% of participants had developed lipodystrophy after 48 weeks treatment, 68% had elevated cholesterol, and 85% had elevated triglycerides.
Many HIV patients being treated with protease inhibitors experience increases in blood cholesterol and triglycerides. It is yet unknown if these increases are related to lipodystrophy or are an entirely separate side-effect, nor is it known what the longer term implications may be. Since high levels of blood cholesterol and triglycerides maintained over long periods may contribute to heart disease.
In a Canadian trial of ritonavir/saquinavir, 11% of patients saw triglyceride levels increase three- to four-fold during treatment. A number of these patients were treated with drugs designed to reduce the levels of lipids in the blood. Gemfibrozil (Lopid) and clofibrate significantly reduced these lipid levels by more than 50% in four out of six cases.
Some physicians conclude that while lipodystrophy is cosmetically undesirable and may cause significant psychological distress, it remains to be seen whether it is a symptom of a serious underlying condition.