The renal tolerance of low-dose adefovir dipivoxil by lamivudine-resistant individuals co-infected with hepatitis B and HIV

Heidi Hannon1, Corinne Isnard Bagnis1, Yves Benhamou1, Hélène Beaufils2, Mark Sullivan3, Carol Brosgart3, Hassan Izzedine1, Thierry Poynard1 and Gilbert Deray1

1Nephrology and Hepatology Departments, Hôpital Pitié Salpétrière and CNRS UPRESA 8087, 2INSERM U423, Hôpital Necker-Enfants Malades, Paris, France and 3Gilead Sciences, Inc., Foster City, CA, USA

Correspondence and offprint requests to: Dr Corinne Isnard Bagnis, Service de Néphrologie, Hôpital Pitié Salpétrière, 83, Boulevard de l’ Hôpital, F-75013, Paris, France. Email: corinne.bagnis{at}psl.ap-hop-paris.fr



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Adefovir (ADV), an orally administered nucleotide analogue active against hepadnaviruses, retroviruses and herpes viruses was shown to be effective in HIV-infected patients, but the prevalence of nephrotoxicity with doses of 60–120 mg/day was considered unacceptable. Recently, lower doses of ADV were shown to be effective for the treatment of HIV-1 patients with chronic lamivudine (LAM)-resistant hepatitis B.

Methods. In a cohort of 35 patients infected with both HIV-1 and LAM-resistant hepatitis B virus, we investigated the renal tolerance of a once-daily dose of ADV 10 mg over 52 weeks. Their mean baseline creatinine clearance was within the normal range (105 ± 3 ml/min/1.73 m2). No patient had significant changes in renal function or electrolyte balance secondary to ADV treatment.

Results. Transient increases in serum creatinine, which resolved by the end of the study were noted in two patients and three developed proteinuria, which was felt to be unrelated to ADV treatment. The cohort's mean serum phosphate level, 2.45 ± 0.09 mg/dl at baseline, did not change significantly under treatment (2.66 ± 0.12 mg/dl at week 52, P = NS).

Conclusions. Our study shows that ADV dosed at 10 mg/day for the treatment of LAM-resistant chronic hepatitis B in patients co-infected with HIV is not associated with renal tubular dysfunction or a significant change in renal function.

Keywords: adefovir dipivoxil; hepatitis B; HIV infection; hypophosphataemia; nephrotoxicity; renal tolerance; tubulopathy



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Lamivudine (LAM)-resistant hepatitis B virus (HBV) is found in ~15–32% of infected patients, with or without HIV-1 infection, after 1 year of LAM therapy [1], in 50% after 2 years and in up to 91% after 4 years. Adefovir dipivoxil (ADV) is active in vivo and in vitro against wild-type and LAM-resistant HBV. Unfortunately, previous studies [2,3] have documented mild to moderate nephrotoxicity with ADV at doses of 60 and 120 mg/day when studied for the treatment of HIV. Proximal renal tubular dysfunction occurred after at least 20 weeks of daily exposure. In an ongoing open-label trial performed in our institution [4], ADV used at 10 mg once daily was shown effective as a treatment of LAM-resistant HBV infection in HIV-1 co-infected patients. In this study, we analysed the same cohort for evidence of nephrotoxicity resulting from ADV, 10 mg once daily.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and study design
In this study we enrolled male and non-pregnant female patients over 18 years who met the following criteria: documented HIV infection; HBV co-infection with HBV DNA detectable in serum (by liquid hybridization assay) at least 6 months before enrollment despite ongoing LAM therapy; inclusion of LAM therapy (150 mg twice daily) in the current anti-retroviral regimen; documented HBV polymerase gene mutations within the YMDD motif; HIV-1 viral load 2.60 log10 copies/ml or less (by Roche Amplicor Monitor, lower limit of quantification 2.30 log10 copies/ml); serum amylase concentration less than 1.5 times the upper limit of normal; neutrophil count >750 x 106/l, platelet count >50 000 x 106/l and haemoglobin concentration >80 g/l; the ability to understand and sign a written consent form. In terms of renal function, the inclusion criteria were: serum creatinine concentration <1.5 mg/dl and a serum phosphate concentration >2.0 mg/dl. Major exclusion criteria were: prior use of ADV; hepatitis delta virus infection; active parasitic, bacterial or viral infections (other than HBV and HIV); a new AIDS-defining event diagnosed <1 month before enrollment; treatment with immunomodulator drugs (interferons, interleukin-2, steroids) in the 4 weeks before enrollment; anti-HBV therapy other than LAM (e.g. interferon alpha, famciclovir, foscarnet, ganciclovir or lobucavir) in the 12 weeks before enrollment; evidence of gastrointestinal malabsorption or chronic nausea or vomiting; malignancies, other than cutaneous Kaposi's sarcoma treated with systemic drugs; decompensated cirrhosis (i.e. Child-Pugh category C); history of heart failure; poor adherence to anti-retroviral therapy; clinically significant neuropathy. The renal exclusion criteria were: clinically significant history of renal dysfunction in the 12 months before enrollment; history of renal failure; concomitant therapy with one or more of aminoglycosides, amphotericin B, cidofovir, cisplatin, intravenous pentamidine, vancomycin, systemic chemotherapeutic agents, D-carnitine or D,L-carnitine or both. All enrolled patients received ADV 10 mg/day for 52 weeks or until withdrawal or mid-course exclusion from the study.

Evaluation of nephrotoxicity
The patients were seen in the outpatient clinic 2 and 4 weeks after the initiation of the study and every 4 weeks thereafter. At each visit, patients were assessed for adverse events and blood tests were performed. Measurements of HBV serological markers and serum HBV DNA, CD4 positive cells and plasma HIV-1 RNA were performed every 12 weeks. The renal parameters evaluated were serum creatinine, urea, electrolytes, calcium, phosphate and bicarbonates. Creatinine clearance was calculated at baseline by the Cockcroft and Gault formula. The protein/creatinine ratio was measured after 52 weeks.

Statistical analysis
The paired t-test method was used to assess change from baseline in biological parameters. The Bonferroni (all-pairwise) method was used to adjust for multiple comparisons. Type I error of 0.05 was considered. Results are expressed as mean ± standard error (SE).



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
At its inception, 35 patients (34 men) were included in the study (mean age 41 ± 2 years), of whom 32 reached week 52: two patients withdrew because of adverse events (diabetes mellitus and increase in serum amylase and lipase levels) and one for personal reasons. Table 1 summarizes the main baseline characteristics of the patients.


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Table 1. Baseline demographic and clinical data

 
Concomitant anti-retroviral therapy
Patients received various combinations of anti-retroviral therapies in addition to LAM. These regimens included protease inhibitors (nelfinavir in three, indinavir in six, ritonavir in combination with saquinavir in four and amprenavir in two) in combination with two nucleoside analogues (didanosine, zalcitabine, zidovudine, stavudine and/or abacavir). In the cohort, 14 patients received a non-nucleoside analogue (nevirapine in seven, efavirenz in seven) in combination with two nucleoside analogues, and six received two or more nucleoside analogues.

Efficacy and general safety (Table 2)
The baseline mean (±SE) of the CD4-positive cell count was 423 ± 35 cells/mm3 and did not change significantly during treatment. Mean serum HBV DNA concentration (Roche Amplicor PCR, lower limit of quantification 2.6 log10 copies/ml) decreased significantly from 8.64 ± 0.08 log10 copies/ml at baseline, to 3.4 ± 0.12 log10 copies/ml at week 24 and to 4.01 ± 0.17 log10 copies/ml at week 48 (P < 0.001). ADV was generally well tolerated, and the clinical status of patients generally improved [4]. The serum concentration of alanine amino transferase increased transiently from a baseline of 103 ± 12 IU/l (peaking between weeks 8 and 20) and decreased thereafter to be significantly lower than baseline by week 48 (74 ± 8 IU/l). Dysuria was not reported by the patients after the initiation of ADV. No significant change in serum sodium and chloride concentrations were observed. A statistically significant increase in serum calcium concentration from 9.16 ± 0.08 (2.29 ± 0.02) to 9.4 ± 0.08 mg/dl (2.35 ± 0.02 mmol/l) was observed at week 52 (P < 0.05), but serum calcium concentration remained within the normal range. The increase in serum calcium levels was not correlated with an increase in serum albumin (mean baseline albumin 4.63 ± 0.08 vs 4.23 ± 0.06 g/dl at week 52, P = NS).


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Table 2. Biological parameters before inclusion and after a 52-week period of ADV treatment

 
Renal function
The mean baseline creatinine clearance (Cockcroft and Gault) was 105 ± 3 ml/min/1.73 m2 and serum creatinine was 0.9 ± 0.02 mg/dl (80.2 ± 2.2 µmol/l). No significant change in serum creatinine was observed after 52 weeks [0.92 ± 0.02 mg/dl (81.0 ± 1.7 µmol/l), P = 0.71]. Patients on ritonavir or indinavir, or both (n = 11), did not have any increase in serum creatinine over time [baseline, 0.94 ± 0.06 mg/dl (83.1 ± 5.0 µmol/l) vs week 52, 0.94 ± 0.03 mg/dl 983.1 ± 2.9 µmol/l), P = NS] with the exception of one patient described below. Indeed, among the 32 patients who completed the first year of the study, two had a serum creatinine increase >20% of the baseline for at least 1 month.

In one patient, the serum creatinine concentration increased from baseline by 0.72 mg/dl (64 µmol/l) at week 28. This patient was being treated concomitantly with efavirenz, abacavir and LAM and had started taking oral acyclovir (200 mg five times a day) 1 week before serum creatinine increased. After discontinuation of acyclovir, serum creatinine returned to normal within 1 week. ADV therapy was not discontinued in this patient. His creatinine value at week 52 was 0.92 mg/dl (81 µmol/l). In another patient, an increase in creatinine by 0.67 mg/dl (59 µmol/l) above baseline was observed at week 32. This patient had cirrhosis, but there was no apparent clinical condition that could be associated with the change in serum creatinine. In this case, serum creatinine returned to baseline within 4 weeks of discontinuing ADV and all anti-retroviral drugs (indinavir, stavudine and LAM). ADV was re-introduced at week 36, with a new combination of anti-retroviral drugs—didanosine, stavudine, LAM, lopinavir and ritonavir—but excluding indinavir. No subsequent increase in serum creatinine was noticed, and the serum creatinine at week 52 was 1.04 mg/dl (92 µmol/l). The transient increase in serum creatinine was attributed to the use of indinavir.

There was no significant increase in serum urea [13.9 ± 0.6 (4.95 ± 0.23) vs 14.9 ± 0.7 mg/dl (5.33 ± 0.25 µmol/l), P = NS]. Serum potassium and bicarbonate concentrations were unchanged (Table 2). Mean baseline serum phosphate concentration was 2.45 ± 0.09 mg/dl (0.79 ± 0.03 mmol/l) (normal 0.8–1.4 mmol/l). No significant change in the level of serum phosphate was observed [mean after 52 weeks, 2.66 ± 0.12 mg/dl (0.86±0.04 mmol/l), P = NS compared with baseline].

Urinalysis
Urinary protein screening (dipstick) initially was negative in all patients. Twenty-four hour measurements were not performed in any patient unless clinically indicated. The mean protein/creatinine ratio, calculated after 52 weeks, was 0.18 ± 0.06. Significant proteinuria, defined as a protein/creatinine ratio >0.2, occurred in three patients of whom two (Patients A and B) had shown significant proteinuria (dipstick screening) during the study. In Patient A, proteinuria was diagnosed on dipstick screening after 24 weeks of treatment and was quantified on a 24-h urine sample (2.34 g/24 h). There was no microscopic haematuria or leucocyturia and no concomitant hypertension. Serum creatinine was 0.89 mg/dl (79 µmol/l) and serum albumin was 3.6 g/dl at that time. The patient was referred to the nephrology department and underwent an uncomplicated renal biopsy. In Patient B, proteinuria together with haematuria were discovered on dipstick screening after 38 weeks. Serum creatinine was 1.01 mg/dl (89 µmol/l), serum albumin 5.4 g/dl. The patient had been treated previously for hypertension. He was referred to the nephrology department and underwent an uncomplicated renal biopsy. A third patient showed a transitory proteinuria after 50 weeks and a significantly elevated protein/creatinine ratio at week 52, but afterwards proteinuria disappeared totally. The patient was still negative for proteinuria at the last follow-up.

Histology
Two of the three patients with proteinuria at week 52 had kidney biopsies after 38 (Patient A) and 40 weeks (Patient B) of ADV treatment. In Patient A (protein/creatinine ratio 1.6), 12 glomeruli were available for light microscopy. All showed diffuse granular deposits in the mesangial areas and irregular deposits along the capillary walls in the sub-epithelial and sub-endothelial spaces. No capillary thrombi or mesangial or epithelial hypercellularity were seen. By immuno-fluorescence, these deposits were composed of IgG, IgM, C1q, C3, Kappa and Lambda. All tubules were normal—without acute necrosis, loss of brush border, cytoplasmic vacuolization, nuclear apoptosis or calcification. This patient also had mixed type III cryoglobulinaemia in the context of concomitant hepatitis C infection (positive anti-hepatitis C antibodies with a negative HCV RNA PCR). The glomerular lesions in this patient were considered to be secondary to the viral infection. In Patient B (protein/creatinine ratio 0.5), the kidney biopsy yielded seven glomeruli of which two were sclerotic. The others seemed lightly hypertrophic with mesangial IgA, IgG, IgM, C3 deposits. Focal interstitial fibrosis with tubular atrophy was noticeable near sclerotic glomeruli. Elsewhere, there was only a moderate irregular flattening of epithelium with loss of brush borders and preservation of some proximal tubules. Cryoglobulinaemia type II was found in his plasma. The third patient, who had diabetes mellitus, did not have a renal biopsy for proteinuria (protein/creatinine ratio 0.98 at week 52) because it resolved spontaneously.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We have shown that, contrary to what has been described with the use of ADV 60 and 120 mg daily in HIV infected patients, treatment with 10 mg of oral ADV once daily for 48 weeks in patients with LAM-resistant chronic hepatitis B and HIV co-infection is not associated with significant renal impairment or proximal tubular dysfunction (based on our study of plasma electrolytes and creatinine). Interestingly, this group of patients is at a particularly high risk of renal dysfunction due to HIV and other viral infections, anti-viral drugs and other conditions associated with chronic hepatitis B.

In three out of 29 patients, significant proteinuria was detected during treatment, but it could not be related to ADV treatment in any of them (based on histopathological findings).

The initial studies of ADV efficacy at doses of 60 and 120 mg/day in the treatment of HIV showed significant resultant renal impairment, and the programme to develop it for that indication was discontinued in 1999. High-dose ADF nephrotoxicity manifested primarily by slowly increasing serum creatinine levels, hypophosphataemia or both. A 0.5 mg/dl (44.2 µmol/l) or more increase above baseline of serum creatinine occurred in 35 and 50% of patients, and hypophosphataemia occurred in 50 and 61% of the patients by 24 and 48 weeks of ADV treatment, respectively. In 88% of those patients, median time to revert the serum creatinine to <0.5 mg/dl (44.2 µmol/l) above baseline was 24 weeks after drug discontinuation [2]. Decrease over time in serum phosphate and time to the resolution of the hypophosphataemia occurred in parallel with the changes in creatinine values. Similar renal function changes were subsequently described by Fisher et al. [3] who studied the effect of 120 mg ADV used for the prevention of CMV retinitis in AIDS patients. In that study, renal dysfunction (one or more of increased serum creatinine >0.5 mg/dl above baseline, serum phosphate <2.0 mg/dl, proteinuria >100 mg/dl or glycosuria >250 g/dl) appeared in 5.8% of the patients by month 6 and in 17% by month 12. The median time to the resolution of nephrotoxicity was 15 weeks; in an estimated 16% of patients, renal dysfunction was not completely resolved by the 41st week, and one patient required dialysis. In addition, a higher incidence of haematuria (dipstick test) was observed in the ADV group, often concomitantly with other renal abnormalities. Proteinuria >2+, identified by dipstick, was present in 33.3% of the ADV patients but also in 17.2% of the placebo patients.

However, the development of the programme to treat chronic hepatitis B with ADV has continued, evaluating the dose of 10 mg once daily [49]. Perillo et al. studied the efficacy and tolerance of lower doses of ADV (5–30 mg) in the treatment of LAM-resistant hepatitis B patients. Out of five patients two had mild increases of serum creatinine, with a follow-up of 13 months. Out of five patients four were liver transplantation recipients who were concurrently receiving numerous other potentially nephrotoxic drugs (such as cyclosporin and tacrolimus); which, therefore, allows making only speculative causality hypotheses. When ADV was given for shorter periods (less than 12 weeks) at higher daily doses (125 or 250 mg), no nephrotoxicity was noted [10].

Histopathological features in ADV nephrotoxicity have been described to our knowledge in only one patient [11]. Glomeruli (n = 35) were normal but cortical tubules revealed severe and diffuse degenerative changes primarily affecting the proximal nephron. There was widespread tubular epithelial simplification with oedema, early fibrosis and a mild interstitial infiltrate consisting predominantly of lymphocytes. By immuno-fluorescence, there was no glomerular or tubulo-interstitial staining, and electron microscopy showed severe widespread alterations of the proximal tubules with striking abnormalities of the mitochondrial size, shape and substructure. It was concluded that ADV nephrotoxicity in this patient may have been associated with the depletion of the mitochondrial DNA of the proximal tubular epithelial cells resulting in the impairment of cellular oxidative respiration.

Indeed, ADV is excreted by a combination of glomerular filtration and active tubular secretion, indicating that its accumulation in proximal tubular cells may play a role in the occurrence of the nephrotoxicity observed during its clinical use [12]. The human organic anion transporter 1 (hOAT 1), localized in the baso-lateral membranes of renal tubular cells, is responsible for the tubular secretion of ADV. Therefore, as the potential for ADV toxicity is increased in various types of mammalian cells that constitutively express hOAT, hOAT1 specific inhibitors may reduce ADV-induced cytotoxicity [13]. It is noteworthy that very little is known about electrolytic and calcium/phosphate abnormalities in HIV patients in general. Therefore, drug-induced tubular dysfunction in HIV patients is not easy to assess. Indeed, tubular damages may also be caused by HIV or HBV infection.

In conclusion, this study demonstrates that the use of 10 mg of ADV once daily for 1 year for the treatment of chronic hepatitis B in patients co-infected with HIV-and LAM-resistant HBV is not associated with significant nephrotoxicity. The use of ADV in patients whose medical conditions put them at high risk of nephrotoxicity or tubular damage definitely must be closely monitored to ensure safe use of this potent anti-viral drug.



   Acknowledgments
 
The work was supported by a grant from SIDACTION and Gilead Sciences.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 20. 3.03
Accepted in revised form: 13. 8.03





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