chronic administration of tenofovir to rhesus …aac.asm.org/content/52/9/3144.full.pdf · november...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2008, p. 3144–3160 Vol. 52, No. 90066-4804/08/$08.00�0 doi:10.1128/AAC.00350-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Chronic Administration of Tenofovir to Rhesus Macaques fromInfancy through Adulthood and Pregnancy: Summary ofPharmacokinetics and Biological and Virological Effects�

Koen K. A. Van Rompay,1* Lucie Durand-Gasselin,2 Laurie L. Brignolo,1 Adrian S. Ray,2Kristina Abel,1 Tomas Cihlar,2 Abigail Spinner,1 Christopher Jerome,3 Joseph Moore,1

Brian P. Kearney,2 Marta L. Marthas,1 Hans Reiser,2 and Norbert Bischofberger2

California National Primate Research Center, University of California, Davis, California 956161; Gilead Sciences, Foster City,California 944042; and Allevia AG, Bern, Switzerland3

Received 12 March 2008/Returned for modification 21 April 2008/Accepted 16 June 2008

The reverse transcriptase (RT) inhibitor tenofovir (TFV) is highly effective in the simian immunodeficiencyvirus (SIV) macaque model of human immunodeficiency virus infection. The current report describes extendedsafety and efficacy data on 32 animals that received prolonged (>1- to 13-year) daily subcutaneous TFVregimens. The likelihood of renal toxicity (proximal renal tubular dysfunction [PRTD]) correlated with plasmadrug concentrations, which depended on the dosage regimen and age-related changes in drug clearance. Belowa threshold area under the concentration-time curve for TFV in plasma of �10 �g � h/ml, an exposureseveralfold higher than that observed in humans treated orally with 300 mg TFV disoproxil fumarate (TDF),prolonged TFV administration was not associated with PRTD based on urinalysis, serum chemistry analyses,bone mineral density, and clinical observations. At low-dose maintenance regimens, plasma TFV concentra-tions and intracellular TFV diphosphate concentrations were similar to or slightly higher than those observedin TDF-treated humans. No new toxicities were identified. The available evidence does not suggest teratogeniceffects of prolonged low-dose TFV treatment; by the age of 10 years, one macaque, on TFV treatment since birth,had produced three offspring that were healthy by all criteria up to the age of 5 years. Despite the presence ofviral variants with a lysine-to-arginine substitution at codon 65 (K65R) of RT in all 28 SIV-infected animals,6 animals suppressed viremia to undetectable levels for as long as 12 years of TFV monotherapy. In conclusion,these findings illustrate the safety and sustained benefits of prolonged TFV-containing regimens throughoutdevelopment from infancy to adulthood, including pregnancy.

Because at present it is not possible to cure human immu-nodeficiency virus (HIV) infection, prolonged treatment withcombinations of anti-HIV drugs is required in order to preventdisease progression. The feasibility of giving HIV-infected per-sons a normal life span through chronic treatment will bedetermined by a number of factors, including compliance andcost, but especially the long-term safety and efficacy of thedrugs.

The nucleotide reverse transcriptase (RT) inhibitor tenofo-vir {TFV; 9-[2-(phosphonomethoxy)propyl]adenine}, in theform of its orally bioavailable prodrug TFV disoproxil fuma-rate (TDF), has become one of the most commonly used anti-HIV drugs due to its favorable efficacy and safety profile, basedon data collected over more than 7 years for HIV-infectedadults (12, 24, 35, 37, 47, 49).

The acyclic nucleoside phosphonates cidofovir, adefovir, andTFV are renally excreted by a combination of glomerular fil-tration and active tubular secretion (14, 15, 18, 38). The effec-tive uptake of acyclic nucleoside phosphonates by organic an-ion transporters in proximal tubules leads to accumulation intubular cells and dose-limiting toxicity in animals (5, 63). Renal

toxicity is usually manifested as renal insufficiency and proxi-mal renal tubular dysfunction (PRTD). During clinical trials ofTDF, the frequency of clinically significant renal changes wasvery low among populations with normal renal values at base-line (and not different from the frequencies seen with otherhighly active antiretroviral therapy regimens [24, 27, 37, 49, 53,69]); furthermore, renal toxicity has been observed infre-quently through continued clinical monitoring as described incase reports and cohort study reports (8, 21, 22, 27, 35, 37, 44,47, 48, 53, 70, 71). Some reports on TDF-treated populationsdescribe small reductions in creatinine clearance that re-mained within the normal range and were thus of uncertainclinical significance (22, 24, 25, 37, 40, 70). Prolonged TDFtreatment of HIV-infected adults was associated with a smalldecrease in bone mineral density (BMD) during the first 48weeks of treatment, but this decrease was nonprogressive(through week 288) and was not associated with any clinicalsymptoms (12, 24). These decreases were associated withmarkers of bone metabolism suggesting a slight increase inbone turnover (full prescribing information for Viread [TDF];available from Gilead Sciences). Some but not all reports havedescribed a similar loss in BMD in children who receivedTDF-containing regimens (23, 26, 30).

Because of many similarities in host physiology and diseasepathogenesis, simian immunodeficiency virus (SIV) infectionof macaques is a well-established and valuable animal model ofHIV infection for testing of many aspects of drug treatment,

* Corresponding author. Mailing address: California National Pri-mate Research Center, University of California, Davis, CA 95616.Phone: (530) 752-5281. Fax: (530) 754-4411. E-mail: [email protected].

� Published ahead of print on 23 June 2008.

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including efficacy and safety (66). Studies with this animalmodel have demonstrated that a once-daily dosage regimen ofTFV was highly efficacious for prophylaxis and therapy and stillhad partial benefit in the presence of viral variants with alysine-to-arginine mutation at codon 65 (K65R) in RT, asso-ciated with approximately fivefold-reduced susceptibility toTFV in vitro (57, 59–62, 65, 67). Previously we reported thatprolonged treatment of macaques with a high dose of TFV (30mg/kg of body weight subcutaneously once daily) resulted inPRTD, a Fanconi-like syndrome characterized by glucosuria,aminoaciduria, hypophosphatemia, and bone pathology; theseeffects were completely or partially reversible following drugwithdrawal or dosage reduction, respectively (63). In contrast,chronic administration of a low dose of TFV (10 mg/kg sub-cutaneously once daily) starting at birth did not seem to beassociated with any adverse health effects within 3 years oftreatment (63).

While some limited early data have been published previ-ously (63), the current report summarizes extended observa-tions on the pharmacokinetics, safety, and efficacy of pro-longed TFV regimens in the macaque model, including duringpregnancy. The new analysis allows us to refine the therapeuticwindow of chronic TFV exposure that balances antiviral effi-cacy with a favorable safety profile. These observations in themacaque model support the long-term treatment of HIV-in-fected humans with TDF-containing regimens.

MATERIALS AND METHODS

Animals, virus inoculation, and TFV administration. All animals were rhesusmacaques (Macaca mulatta) from the type D retrovirus-free and simian T-cell-lymphotropic virus type 1-free colony at the California National Primate Re-search Center (CNPRC). Newborn macaques were hand reared in a primatenursery in accordance with the standards of the American Association for Ac-creditation of Laboratory Animal Care. We adhered strictly to the Guide for theCare and Use of Laboratory Animals, prepared by the Committee on Care andUse of Laboratory Animals of the Institute of Laboratory Resources, NationalResearch Council (46). When necessary, animals were immobilized with keta-mine HCl (Parke-Davis, Morris Plains, NJ) at 10 mg/kg injected intramuscularly.Blood samples were collected regularly for monitoring viral and immunologicparameters as described previously (65). Complete blood counts were performedon EDTA-anticoagulated blood samples. Samples were analyzed using an auto-mated electronic cell counter (Baker 9000; Serono Baker Diagnostics) and, fromNovember 2002 onward, a Pentra 60C� analyzer (ABX Diagnostics); differentialcell counts were determined manually. Three-color and four-color flow cytom-etry techniques (including CD3, CD4, CD8, and CD20) were performed asdescribed previously (58–60, 65).

The animals described in this report included all 32 animals that were givensubcutaneous TFV treatment for �1 year in experiments starting from 1995onward, and their offspring. Twenty-eight of these 32 animals were infected withSIV or RT-SHIV (a chimeric SIV containing HIV type 1 [HIV-1] RT) either atbirth or at a juvenile age. More-detailed descriptions of the viral inoculum stocksand the early virological and immunological data have been published previouslyfor all of these animals (58–60, 65). Plasma viral RNA concentrations in theseanimals were determined by a quantitative branched-chain DNA (bDNA) assayfor SIV-infected animals or by a real-time reverse transcription-PCR (RT-PCR)assay for SIV gag, as described previously (58, 65).

TFV powder (supplied by Gilead Sciences, Inc., Foster City, CA) was sus-pended in distilled water, dissolved by addition of NaOH to a final pH of 7.0 ata final concentration of 40 or 60 mg/ml, and filter sterilized (pore size, 0.2 �m;Nalgene, Rochester, NY). Although it is very stable at ambient temperatures, theTFV solution was stored at 4°C, and new stock solutions were prepared every fewmonths. TFV was administered once per day subcutaneously into the back of theanimal, at dosage regimens described below. Unless indicated otherwise, TFVdosages were adjusted weekly according to body weight.

As explained in Results, following the onset of renal toxicity and hypophos-phatemia, some animals were administered a phosphate supplement once or

twice daily; depending on the animal’s weight, each dose consisted of 1 or 2packages of Neutra-Phos (250 mg phosphorus, 164 mg sodium, and 278 mgpotassium per package; Baker Norton Pharmaceuticals, Miami, FL) dissolved ina 100-ml solution of Tang (Kraft Foods). A pediatric multivitamin tablet with 200mg calcium (CentrumKids Extra Calcium; Lederle, Madison, NJ) and/or one-quarter of an Amino Fuel tablet (Twin Laboratories Inc., Ronkonkoma, NY)with amino acids was administered to some animals once daily.

Serum chemistry panels. Standard chemistry panels (including sodium, potas-sium, chloride, calcium, total CO2, anion gap, calcium, phosphorus, creatinine,blood urea nitrogen, glucose, alanine aminotransferase, alkaline phosphatase,total protein, albumin, gamma-glutamyltransferase, creatine phosphokinase, as-partate transaminase, total bilirubin, lactate dehydrogenase, cholesterol, andtriglycerides) were performed with the Dacos (Coulter Electronics, Hialeah, FL)or the Hitachi 717 (Roche Biomedical, Indianapolis, IN) system. Serum sampleswere generally collected from animals after overnight fasting.

Urine analysis. Urine was collected by cystocentesis. Urine was analyzed forspecific gravity by a refractometer; the pH and the presence of protein, glucose,ketone, bilirubin, and occult blood were determined using Multistix strips (BayerCorporation, Elkhart, IN). Urine sediments were examined microscopically.

Radiographs and DXA. Radiographs were taken with a Thompson CGR X-rayunit (model SPG 515S) with the following settings: 100 mA, 0.10 s, 46 to 54 kVp(kilovoltpeak). Dual-energy X-ray absorptiometry (DXA) scans were performedwith a Norland Eclipse compact DXA system. Animals were placed in standard-ized positions for scans. The bone mineral content (BMC), bone mineral area(BMA), and BMD (calculated as BMC/BMA) were determined for each site.Machine software calculates BMC based on the X-ray absorption characteristicsof the tissue and determines BMA by calculating the projected bone area usingan edge detection algorithm to find the bone margins within the DXA image.Measurement sites included lumbar vertebrae 2 to 4 (L2 to L4), distal radius andulna (DR�U), femoral neck, and global proximal femur.

TFV pharmacokinetics. Blood was collected by venipuncture at various times(0, 0.5, 1, 2, 4, 8, and 24 h; in some experiments, blood was also collected at 6 h)after subcutaneous TFV administration and was spun for 10 min at 900 � g toseparate the plasma or serum from the cells. Plasma and serum were immedi-ately stored at �70°C. At selected time points, peripheral blood mononuclearcells (PBMC) were isolated by Ficoll gradient separation (lymphocyte separationmedium; MP Biomedicals, Aurora, OH) and washed in a cold (�4°C) 0.9% NaClsolution. To lyse contaminating red blood cells, previously published techniqueswere used (20). Briefly, the cells were incubated for 2 min with 2 ml of ammo-nium salt solution (3.5 g ammonium chloride and 36 mg ammonium carbonateper 500 ml water) and then washed twice with cold 0.9% NaCl, and the cellpellets were stored at �70°C until analysis.

Concentrations of TFV in plasma or serum were determined either by avalidated high-performance liquid chromatography (HPLC) method with fluo-rescence derivatization with a limit of quantitation (LOQ) of 25 ng/ml (54) or bya validated HPLC coupled to a positive-ion electrospray tandem mass spectrom-etry (LC–MS-MS) detection method with an LOQ of 3.7 ng/ml (63).

Plasma was prepared by protein precipitation by the addition of 2 equivalentsof acetonitrile containing 0.2% formic acid and 150 nM adefovir (Gilead Sci-ences), which was chosen as an internal standard. Following filtration, sampleswere dried and reconstituted in mobile phase A (0.2% formate in water) beforeanalysis using LC–MS-MS. Chromatography was performed on a Synergi HydroRP-80 Å 4-�m, 75- by 2.0-mm column (Phenomenex, Torrance, CA). Analyticalseparation was accomplished using 0.2% formate as a buffer and a multistagegradient between 1% and 95% acetonitrile over 3.5 min by maintaining a flowrate of 250 �l/min with a Shimadzu LC-20AD tertiary pump system (ShimadzuScientific Instruments, Columbia, MD). The column was reequilibrated for 1 minbefore injection of the next sample. Parent/daughter mass-to-charge (m/z) tran-sitions of 288.2/176.1 and 274.1/162.0 (resolution in atomic mass units) for TFVand adefovir (internal standard) were monitored on an API-4000 triple-quadru-pole mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA). Con-centrations were determined based on calibration curves (1/x, weighted) withlinearity in excess of an r2 of 0.99 and with a typical LOQ of 3.7 ng/ml.

Pharmacokinetic parameters were derived by noncompartmental analysis us-ing WinNonlin, version 2.1, 3.1, or 5.0.1 (Pharsight Corporation, Mountain View,CA). While the TFV bioavailability (F) after subcutaneous administration isexpected to be �100%, all clearance values in this report refer to apparentclearance (CL/F) rather than absolute clearance (CL).

Quantitation of intracellular TFV-DP. PBMC samples were lysed in 0.5 ml ofcold 70% methanol. Following centrifugation, cell debris was separated from thePBMC extract and used to evaluate the exact number of cells by using a validatedbiochemical assay as previously described (6). A 225-�l aliquot of lysed PBMCwas used for direct quantitation of TFV diphosphate (TFV-DP). The PBMC

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extract was dried and resuspended before analysis in 1 mM phosphate buffercontaining 225 nM 2-chloro-ATP (Sigma-Aldrich, St. Louis, MO), chosen as aninternal standard. Intracellular TFV-DP concentrations were determined byLC–MS-MS using a method derived from previously described ion-pairing andlow-flow nucleotide detection methods (50, 68). Chromatography was performedon an Acquity BEH-C18 1.7-�m, 2.1- by 5.0-mm column with additional use of aVanGuard precolumn (Waters, Wexford, Ireland). Analytical separation wasaccomplished using 3 mM formate and 10 mM 1,5-dimethylhexylamine (Sigma-Aldrich, St. Louis, MO) as an ion-pairing buffer and a multistage gradientbetween 5% and 75% acetonitrile over 6.0 min by maintaining a flow rate of 100�l/min with a Shimadzu LC-20AD tertiary pump system. The column waswashed for 1 min, followed by reequilibration for 2.5 min before injection of thenext sample. Parent/daughter m/z transitions of 448.1/176.1 and 542.0/169.9 (res-olution in atomic mass units) for TFV-DP and 2-chloro-ATP (internal standard)were monitored on an API-4000 triple-quadrupole mass spectrometer. Concen-trations were determined based on calibration curves (1/x, weighted) with lin-earity in excess of an r2 of 0.99 and with a typical lower limit of quantification of39.1 fmol/sample. This value was then divided by the number of cells per sample(typically �1 million) to obtain the final concentration in femtomoles per millioncells.

Measurement of SIV-specific cell-mediated immune responses. Flow cytom-etry assays with intracellular cytokine staining were performed according tostandard protocols using 1 � 106 cells per stimulus in RPMI with 10% heat-inactivated fetal bovine serum. A peptide pool of SIVmac239gag p27 was used at5 �g/ml; aldrithiol-2 (AT-2)-inactivated SIVmac239 was used at 300 ng/ml.Cultures were stimulated with Staphylococcus enterotoxin B (200 ng/ml) as apositive control. Negative-control cultures consisted of medium only. Costimu-latory antibodies to CD49d and CD28 were added, and cultures were incubatedat 37°C under 5% CO2 for 6 h (10). Brefeldin A (10 �g) was added 1 h after thestart of the incubation. Monensin was used instead of brefeldin A to assess thecytotoxic function of T cells by measuring the surface expression of CD107a/b(7). Data were acquired (300,000 lymphocyte events) on a FACS ARIA instru-ment (Becton Dickinson) and analyzed using FlowJo software, version 8.1 (Tree-Star, Ashland, OR). Data were reported as frequencies of positive CD4� orCD8� T cells and were considered positive if the value was �2-fold the medium-only value.

Statistical analysis. Statistical analyses were performed with Prism 4 for Macand Instat 3 (GraphPad Software Inc., San Diego, CA). Statistical analysis ofdisease-free survival was done using a log rank test. A P value of �0.05 wasconsidered statistically significant.

RESULTS

Physiologic age-related changes in systemic TFV clearance.To explore normal age-related changes in systemic TFV CL/F,a single dose of TFV (10 or 30 mg/kg) was administered sub-cutaneously to 23 uninfected animals of different ages, andplasma samples were collected over a 24-h period; 2 animalswere given two doses of TFV (10 mg/kg subcutaneously) 19months apart. TFV clearance was low at birth but then in-creased rapidly over the first year of life to maximum levels(�1,000 ml/h/kg). When juvenile animals matured into adult-hood as assessed by both age and weight, TFV clearance de-creased to reach normal levels of approximately 400 to 750ml/h/kg (Fig. 1A and B). For comparison, the renal blood flowand glomerular filtration rate in rhesus macaques of 5 kg havebeen reported to be 1,650 ml/h/kg and 125 ml/h/kg, respectively(17).

Pharmacokinetics and toxicity during prolonged TFV treat-ment. (i) Animal population. Thirty-two animals that were partof several different studies had in common that they receiveduninterrupted daily administration of TFV for at least 1 year.While early data on 11 animals have been reported previously(63), the summary in Table 1 includes additional animals andextended data. While 4 of the 32 animals were uninfected andwere used to study only the safety of prolonged TFV admin-istration, the remaining 28 animals were from studies that

investigated the long-term therapeutic efficacy of TFV mono-therapy against SIV or RT-SHIV infection.

(ii) General overview of dose-related toxicity and pharma-cokinetics. In the current follow-up studies, the primary dose-related toxicity of prolonged TFV treatment continued to bethe previously described renal toxicity, namely, PRTD, char-acterized by glucosuria and changes in the levels of certainserum markers (especially hypophosphatemia and elevated to-tal alkaline phosphatase levels). In severe cases, PRTD led tobone pathology (63). Serum creatinine and potassium concen-trations were less-sensitive markers of renal toxicity, since forsome animals, these levels were within the range of those ofuntreated animals (creatinine, �1.4 mg/dl [within or equiva-lent to the mean � 2 standard deviations]; potassium, �2.8mmol/liter) despite significant glucosuria and hypophos-phatemia. Other serum parameters such as liver enzymes andfasting levels of glucose, triglycerides, and cholesterol wereindistinguishable from those of age-matched untreated ani-mals (data not shown); any aberrations of these serum markersin individual animals could be explained by the presence ofSIV-associated pathology in animals that progressed to AIDS(e.g., opportunistic infections resulting in diarrhea, septicemia,etc.).

For the purpose of further analyses, the onset of detectablerenal toxicity (PRTD) was defined as the first observation ofglucosuria (in the absence of hyperglycemia) and/or hypophos-phatemia. Based on the range of values seen in age-matchedcontrol animals, renally induced hypophosphatemia for ani-mals up to the age of �5 years was defined as serum phospho-rus concentrations of �4 mg/dl at two consecutive time pointsor �3 mg/dl at one time point in the absence of major gastro-enteric problems (e.g., due to AIDS) that could suggest re-duced intestinal absorption of phosphate; for adult animalsfrom �5 years onwards, only serum phosphorus concentra-tions of �2.5 mg/dl were considered significant indicators ofhypophosphatemia.

All available longitudinal and cross-sectional pharmacoki-netic data from long-term TFV-treated animals were analyzedin order to determine whether the TFV clearance and/or thearea under the plasma concentration-versus-time curve (AUC)were predictive of the presence or future development ofPRTD (glucosuria and/or hypophosphatemia). For newbornand infant macaques, low TFV clearance and high plasmaAUCs were not associated with detectable PRTD. For adultand juvenile macaques (all �10 months old), the presence ofPRTD at the time of a pharmacokinetic study was associatedwith reduced TFV clearance (�400 ml/h/kg) in comparison tothat of age-matched control animals (Fig. 1C and D). Thecombined analysis of TFV clearance and AUCs provided use-ful insights into the pathogenesis of PRTD during the differentTFV regimens. As indicated in Fig. 2, the combination of lowAUCs (�20 �g � h/ml) and normal TFV clearance (�400 ml/h/kg) was not associated with detectable PRTD, even for ani-mals that were treated for more than 9 years (e.g., animal31122 [Fig. 2, quadrant A]). Animals that had initial TFVclearances within the normal range (�400 ml/h/kg) but higherplasma AUCs (�20 �g � h/ml or higher), either due to higherdosage regimens (20 or 30 mg/kg subcutaneously once daily) ordue to a gradual age-related decrease in clearance (as ex-plained above), were in an intermediate stage (Fig. 2, quadrant

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B), since the prolonged exposure eventually led to the occur-rence of PRTD concomitantly with the observation of reducedTFV clearance (�400 ml/h/kg [Fig. 2, quadrant C]). Followingthe detection of PRTD, empirical TFV dosage reductions wereimplemented to bring AUCs to 10 to 20 �g � h/ml; althoughthis led to improvement, glucosuria and hypophosphatemia didnot always resolve completely, and prolonged treatment withthese reduced regimens generally led to a further decrease inTFV clearance with relapses of the glucosuria and hypophos-phatemia, which then necessitated further dosage reductions.

As our knowledge improved over time, we learned that (i)lower AUC target values of �5 to 10 �g � h/ml were moreappropriate for minimizing the risk of toxicity while maintain-ing antiviral activity (see discussions below, e.g., of animals32186, 30577, 33088, and 33091), and (ii) for TFV dosageregimens that were adjusted weekly based on weight, theweight- and age-related physiologic decrease in clearance (Fig.1A and B) automatically predisposed growing animals tohigher plasma AUCs, which then promoted or aggravated re-nal toxicity. Accordingly, it was then decided to maintain all

FIG. 1. Changes in TFV clearance: effects of age and dosage regimens. Twenty-four-hour pharmacokinetic studies with subcutaneous admin-istration of TFV were performed. (A and B) Weight- and age-related changes in TFV CL/F, respectively, following a single-dose administrationof 10 or 30 mg/kg (subcutaneously) to 23 healthy, uninfected macaques of different ages; 2 of these 23 animals (male 31007 and female 31456) weregiven two doses of TFV (10 mg/kg,� subcutaneously) 19 months apart. (C) TFV CL/F in animals that received prolonged TFV regimens (oncedaily subcutaneously; regimens are outlined in Table 1). Pharmacokinetic data were collected either from animals that never showed PRTD (blue)or from animals prior to (red) or after (black) the development of PRTD. Animal 33091 had glucosuria at a single time point when the TFVclearance was 400 ml/h/kg, but this disappeared when clearance increased again after dosage reduction. (D) An overlay of graphs B and C withanimals grouped according to the presence of PRTD demonstrates that animals that received chronic TFV treatment and never showed signs ofPRTD throughout their observation periods had apparent TFV clearances indistinguishable from those of animals that received a single dose.Based on the available data, the horizontal dotted line indicates the arbitrary cutoff value of reduced TFV clearance (�400 ml/h/kg) that wasassociated with PRTD in juvenile and adult animals.



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2904

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remaining TFV-treated animals (including those without anyevidence of PRTD) on a fixed absolute dose (in mg of TFV)instead of a weight-adjusted dose (Table 1).

For clarity of further presentation, animals are stratifiedaccording to their initial dosage regimen and the occurrence ofPRTD; selected animals are described in more detail to doc-ument the model presented in Fig. 2.

(iii) Prolonged low-dose TFV regimens (always <10 mg/kg;n � 3). Three infant macaques were started on a 10-mg/kg(once daily subcutaneously) TFV regimen (Table 1). SIV-in-fected animal 31042 started receiving TFV at the age of 3weeks, developed K65R RT mutants, had persistent viremia,and eventually had to be euthanized with AIDS at the age of3.5 years (without any evidence of TFV-induced toxicity [63]).The other two animals (male 31121 and female 31122) wereuninfected and were started on a 10-mg/kg regimen within thefirst 2 days after birth. At the age of 3.5 years, when plasmaTFV AUCs increased to �20 �g � h/ml, animal 31121 devel-oped glucosuria. Although gradual dosage reductions (to ob-tain AUCs of 10 to 20 �g � h/ml) produced an initial improve-ment in the markers, eventually the glucosuria became morefrequent and severe (�1,000 mg/dl), and there was a gradualincrease in serum creatinine concentrations (peak, 2.2 mg/dl)and a gradual decrease in serum phosphorus concentrations(nadir, 1.7 g/dl). Accordingly, at the age of 75 months, TFVtreatment was completely withdrawn, which then led to a slow

improvement and stabilization of serum phosphorus (2.1 to 4.3mg/dl) and creatinine (2.0 to 2.2 mg/dl) levels, and the glucos-uria became low (�100 mg/dl) and less frequent. Animal 31121continued to gain weight (currently 11 kg at 10 years), andexcept for the laboratory parameters indicative of reducedrenal function (i.e., increased creatinine levels), is clinicallyhealthy. Throughout this whole observation period, animal31121 received the routine CNPRC primate diet (without anyextra mineral, vitamin, or amino acid dietary supplements).

Female animal 31122 never had detectable glucosuria duringher 10 years of daily TFV treatment. Creatinine concentrationsremained low (�0.8 mg/dl), and serum phosphorus levels andall other serum chemistry parameters remained within theexpected levels for age-matched animals. Serum phosphorusconcentrations decreased transiently (to nadirs of 2.8 to 2.9mg/dl) during consecutive pregnancies (see below), but thesechanges were within the range of values observed for untreatedpregnant animals. Animal 31122 remains healthy at the age of10 years.

(iv) Prolonged TFV treatment with regimens including >10mg/kg (n � 29). Twenty-nine animals had treatment periodsranging from 7 weeks to 25 months where the daily subcuta-neous dose of TFV was 20 or 30 mg/kg. Based on the occur-rence of PRTD, these 29 animals can be separated into twogroups.

FIG. 2. Correlation between apparent TFV clearance, plasma AUCs, and the development of PRTD. Animals had been on stable subcutaneousTFV dosage regimens for at least 4 months at the time of the pharmacokinetic studies. Symbols indicating whether PRTD (glucosuria and/orhypophosphatemia) occurred refer to the time frame after the pharmacokinetics study was performed. While some animals were previously onhigher-dosage regimens (see Table 1), the dosages indicated refer to the time of the pharmacokinetic studies. Because clearance is calculated asdose/AUC, hyperbolas are predicted at a specific dose. The quadrants define where differences and changes in TFV CL/F are physiologic (i.e., dueto individual variation and the effect of aging) versus pathological (i.e., nephrotoxicity). The available data suggest that the cutoff values for thedifferent quadrants are approximately AUCs of 20 �g � h/ml and clearance values of 400 ml/h/kg. Quadrant A is the zone where no PRTD wasobserved; quadrant B is a pre-PRTD stage; quadrants C and D area are associated with clinical PRTD. Glucosuria was detected once for animal33091 after the pharmacokinetic values were at the intersection of these quadrants; the glucosuria resolved following dosage reduction (which wasassociated with an increase in TFV clearance [quadrant A]). (Inset) Empirical model for the pathogenesis of PRTD during high-dose TFVregimens. At high TFV exposures (quadrant B), the gradual development of PRTD reduces TFV clearance; this reduced clearance, by furtherincreasing drug exposure (i.e., AUCs), aggravates renal toxicity and drives the animals’ pharmacokinetic parameters further and further intoquadrant C; only following drastic dosage reductions can values move from quadrant C toward quadrant D.



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(a) Animals without persistent PRTD (n � 7). Seven ani-mals receiving a regimen of �10 mg/kg (Table 1) either neverdeveloped PRTD (n � 4) or had only a single time point atwhich a trace or small amount of glucose (100 to 250 mg/dl)was detected in the urine (n � 3). Following dosage reduction,glucose was absent in the urine at later time points for thesethree animals. Six of the seven animals without persistentPRTD were started on a daily 30-mg/kg regimen that wasreduced within 2 months to 20 mg/kg and then further reducedto 10 mg/kg. The seventh animal (30576) had been on a 20-mg/kg regimen for 15 months without developing detectablePRTD; this absence of renal toxicity is probably due to arelatively high TFV clearance in this animal (1,080 ml/h/kg),leading to plasma AUCs (18.8 �g � h/ml) lower than those foreight other animals that developed PRTD within 3 to 14months of treatment with a 20-mg/kg regimen (Table 1; Fig. 2).All serum chemistry values (including phosphorus, creatinine,and alkaline phosphatase) of these seven animals were withinthe range of age-matched untreated control animals. Theyreceived a regular diet (i.e., without any special mineral, vita-min, or amino acid dietary supplements) throughout their ob-servation period.

(b) Animals with PRTD (n � 22). The other 22 animals thathad been on 20- to 30-mg/kg TFV regimens for extendedperiods developed PRTD (glucosuria and/or hypophos-phatemia) (Table 1).

Of these 22 animals, 6 had evidence of possible PRTD-related moderate hypokalemia (i.e., at least one time pointwhen serum potassium concentrations were �2.8 mmol/liter inthe absence of other obvious explanations such as diarrhea);severe hypokalemia (serum potassium concentrations of �2mmol/liter) was never observed. The serum creatinine concen-tration was an unreliable marker for renal toxicity. For 18 ofthese 22 animals with PRTD, serum creatinine levels remainedwithin the normal range (�1.4 mg/dl) throughout their obser-vation periods; this included animals that had persistent hy-pophosphatemia and/or glucosuria for as long as 5 years. Thisfinding suggests that for these animals, the renal pathology wasmostly limited to the proximal renal tubuli. For four animals,serum creatinine concentrations increased to �1.4 mg/dl, butthe onset of the creatinine increase was unpredictable (7months to 4 years after the onset of hypophosphatemia orglucosuria). Peak creatinine levels ranged from 2.9 to 3.6 mg/dl; increases in creatinine levels were generally gradual andslow. An exception was animal 32186, for which creatininelevels increased suddenly (from 1.4 to 2.1 mg/dl within 6weeks) when this chronically infected animal was depleted ofCD8� cells by the administration of a monoclonal antibody(which was associated with a transient increase in plasma vire-mia) (65); it is possible that immune complexes that wereformed during this CD8� cell depletion experiment may havesuddenly aggravated the development of glomerulonephritis.

Following the occurrence of glucosuria, hypophosphatemia,or increased creatinine levels, sequential dosage reductions (toeventually obtain AUCs of �10 �g � h/ml) and a change to aconstant, absolute dose (irrespective of body weight, as ex-plained above) helped stabilize and/or improve the serumphosphorus and creatinine concentrations. In some animals,such low-dose maintenance regimens (AUCs, �10 �g � h/ml)led to improvement (i.e., an increase in TFV clearance and the

disappearance of glucosuria [Fig. 1C; Table 1, animals 29046,32186, and 30577]). None of these animals with elevated cre-atinine levels had outward symptoms of renal insufficiency.

Based on clinical observations described previously (63),eight of the animals (29045, 29046, 29276, 30007, 30162, 30577,32186, and 33109) for which serum phosphorus concentrationsdid not increase sufficiently shortly after the initial TFV dosagereductions benefited from receiving dietary phosphate supple-ments (Neutra-Phos). Four long-term-treated animals (29045,29046, 29276, and 30577) with poor fur quality (alopecia) werealso given daily amino acid supplements (Amino Fuel) to offsetthe urinary loss of amino acids during PRTD; the initiation ofthese amino acid supplements was sometimes associated withrapid improvement of these animals’ fur.

Effect of prolonged TFV administration on BMD. DXAscans were available for six animals with clinical PRTD (glu-cosuria and/or hypophosphatemia) (Fig. 3). As described pre-viously (63), three of these animals (29045, 29046, 29276) hadreceived a prolonged high-dose TFV regimen (30 mg/kg sub-cutaneously once daily) since birth and showed growth retar-dation with significant radiographic evidence of bone mineral-ization defects before dosage reductions and subsequentdietary supplements were implemented. Despite improvement,the BMDs and BMCs of these three animals remained belowthe range of those of normal adult animals but were not asso-ciated with any overt symptoms; no fractures have been ob-served. Two animals with persistent PRTD for which dosagereductions and dietary supplementation had been initiated ear-lier to prevent major adverse effects displayed either normalBMD (animal 32186) or reduced BMD (animal 30577) after 73or 91 months of TFV administration, respectively. Animal31121 (for which TFV treatment was stopped after �6 yearsdue to PRTD) had BMD within the normal range throughoutthe observation period of 9 years (Fig. 3).

Three animals (31122, 33088, and 33091) (Table 1) thatreceived low-dose maintenance regimens of TFV and had noPRTD or only a single observation of glucosuria throughout 6to 10 years of TFV treatment had total body weights, as well asBMDs and BMCs for L2 to L4 and DR�U, within the rangeof those of age- and sex-matched control animals (Fig. 3). ForTFV-treated female animal 31122, transient decreases in BMDand BMC were observed on DXA scans performed shortlyafter pregnancies; small decreases in BMD in the forearms andlumbar spines of uninfected and untreated pregnant humanshave also been described (43).

Effect of prolonged low-dose TFV treatment during preg-nancy. Uninfected female macaque 31122 was started on pro-longed TFV treatment (10 mg/kg subcutaneously once daily) atthe age of 1 day (Table 1). She was housed with uninfected,TFV-treated male animal 31121. At the age of approximately4 years, she delivered an infant, which died of neonatal septi-cemia. At the age of 5 years and 4 months, she delivered ahealthy male infant (animal 35316). Starting at the age of 7months, animal 35316 has been living in our outdoor colonywith other animals to promote physical activity and social in-teractions; he has developed normally by all criteria (includingbehavior, weight, serum chemistry analyses, urinalysis [no glu-cosuria], radiographs, and BMD [Fig. 3]) throughout his ob-servation period, currently 5 years. When animal 31122 wasalmost 6 years old, a third pregnancy was lost due to a placental



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hemorrhage on gestational day 72, necessitating a cesareansection; the dead fetus was macroscopically normal, and thecause of death was therefore most likely the placental hemor-rhage. At the age of approximately 8 years, animal 31122became pregnant by a different, uninfected, untreated male.Regular ultrasound monitoring revealed normal fetal develop-ment but a partial placenta previa; to prevent any complica-tions, a scheduled cesarean section was performed on gesta-tional day 156, with delivery of a healthy male infant (animal37770; birth weight, 505 g). This infant, which was raised by afoster mother, is currently 22 months old and shows normaldevelopment by all criteria (as described for animal 35316above). A fifth pregnancy at the age of 9 years resulted in fetalloss at approximately gestational day 53. At the age of 10years, a sixth pregnancy, fathered again by animal 31121,resulted in a healthy male infant that was delivered by ce-sarean section (due to a partial placenta previa) on esti-mated gestational day 152.

As a comparison, the rate of prenatal mortality in rhesusmacaques at the CNPRC is 17% (with the highest rate of lossduring early gestation [gestational days 18 to 70]) (31), andtherefore the loss of pregnancy in this animal was not deemedto be TFV related. Four pharmacokinetic studies that wereperformed on animal 31122 between the ages of 3 and 9 yearsindicate that during these consecutive pregnancies, maternalexposure to TFV (plasma AUCs) ranged from 6 to 19�g � h/ml (Table 1; Fig. 2).

Sustained antiviral efficacy of prolonged TFV treatment.The 28 SIV-infected, TFV-treated animals were from severalstudies where untreated animals had persistently high viremialevels and all developed AIDS (generally within 4 months foranimals infected shortly after birth; within 1 to 2 years foranimals infected as juveniles) (58–60, 64, 65). TFV therapy

initiated early during the infection course generally inducedrapid suppression of viremia. Although prolonged TFV ther-apy always resulted in the emergence of viral mutants with aK65R mutation in RT, the TFV-treated animals had signifi-cantly improved disease-free survival compared to untreatedanimals in all these studies (59, 60, 64, 65) (Fig. 4). Of the 28long-term TFV-treated animals infected with K65R viral mu-tants, 20 had persistent viremia and eventually developedAIDS while on treatment; 1 animal (29003 [Table 1]) becameviremic and developed AIDS after TFV treatment was discon-tinued to reduce toxicity; 1 viremic animal (27999) was AIDSfree but was euthanized because of its skeletal lesions, as de-scribed previously (63). The remaining six animals have beenable to suppress plasma viremia to undetectable levels for 6 to12 years without progression toward AIDS. Whether an animalhad persistent or undetectable viremia did not correlate withthe TFV dosage regimen, the plasma TFV exposure levels, thetiming of the first detection of K65R viral mutants, or thepresence of other mutations in RT. While one animal withundetectable viremia (29045) was eventually lost from thestudy after 7 years of TFV treatment due to an unrelated cause(self-injurious behavior), five infected animals remainedhealthy and had very low or undetectable viremia (less than thecutoff value of 125 copies/ml by a bDNA assay) and normalCD4� and CD8� T-lymphocyte counts and CD4/CD8 lympho-cyte ratios after their respective TFV treatment periods (12years for animal 29276, 8 years for animal 30577, 7 years foranimal 32186, and 6 years for animals 33088 and 33091). Infour of these five animals with undetectable viremia by con-ventional diagnostics (29276, 30577, 32186, and 33088), novirus could be detected in plasma even by the more sensitivereal-time RT-PCR assay (cutoff, 10 RNA copies per ml ofplasma). The fifth animal (33091) had undetectable viremia as

FIG. 3. DXA scan evaluation of bones in long-term TFV-treated animals. Details on the TFV dosage regimens are provided in Table 1. DXAscans were performed on the femoral neck, global proximal femur, DR�U, and lumbar vertebrae (L2 to L4). Although all these locations gavesimilar results, the BMDs of lumbar vertebrae are shown because they had less variability (among both treated and untreated animals) and weretherefore more useful for comparison of the TFV-treated animals. The untreated female macaques either were nulliparous or were tested at least14 months after their most recent infant had been weaned. Except for those animals that had been on prolonged high-dose TFV regimens and hadbone mineralization defects before the dosage was reduced (29045, 29046, 29276, and 30577), the TFV-treated animals had BMDs similar to thoseof age- and sex-matched control animals. M and F indicate male and female, respectively.



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measured by the bDNA assay (�125 copies per ml) in 8 of the10 samples collected from 10 months of treatment onward buthad 2 intermittent samples with detectable low-level viremia bythe SIV bDNA assay (128 and 678 SIV RNA copies/ml at 20and 47 months of treatment, respectively); plasma collectedafter 66 months of TFV treatment had a copy number of290/ml according to the real-time RT-PCR assay.

In a subset of these six animals with undetectable viremia,transient CD8� cell depletion (via injection of the anti-CD8monoclonal antibody cM-T807) (n � 4) or short-term inter-ruption of TFV treatment (7 to 9 weeks) (n � 4), describedpreviously, had led to a transient increase in viremia (58, 65).Thus, the drug concentrations in these animals, even after thedosage reductions (Table 1), were associated with antiviralactivity.

PBMC of the five surviving SIV-infected animals with verylow or undetectable viremia were tested for the presence ofSIV-specific cell-mediated immune responses using flow cy-tometry assays for intracellular staining of three cytokines:interleukin 2 (IL-2), gamma interferon (IFN-), and tumor

necrosis factor alpha (TNF-). All five animals had detectableSIV-specific CD4� T lymphocytes; four of the five animals hadSIV-specific cytokine-secreting CD8� T lymphocytes in pe-ripheral blood (Table 2). There was no obvious correlationbetween the patterns of cytokine secretion and the duration ofTFV treatment.

These TFV-treated animals continue to be monitored indef-initely.

Intracellular TFV-DP concentrations during single-dose orprolonged TFV treatment. During a 24-h pharmacokineticsstudy, multiple PBMC samples were collected from the sevensurviving long-term TFV-treated animals (doses, 0.4 to 9.1mg/kg subcutaneously) and from four single-dosed juvenilemacaques (4 mg/kg subcutaneously) for the measurement ofintracellular concentrations of the active metabolite, TFV-DP.For the animals on prolonged dosing with TFV, intracellularTFV-DP concentrations varied little over the 24-h samplingperiod (coefficient of variation, �20%) (data not shown), in-dicating the long half-life of TFV-DP in PBMC; TFV-DPconcentrations measured 24 h after the daily drug dose ranged

FIG. 4. Effect of long-term TFV treatment on disease progression in SIV-infected macaques. Disease-free survival curves are presented for allstudies that allowed evaluation of the effect of prolonged TFV treatment on SIV disease progression. Data from separately performed studies werepooled for the analysis if all input parameters (age of animals, virus isolate, dose and route of virus inoculation) were very similar. On each graph,the survival curves of TFV-treated and untreated animals were compared using the log rank test. Long-term survivors that are discussed in Resultsare indicated by arrows. (A) Newborn macaques were inoculated orally with a high dose of SIVmac251, and TFV treatment was started on fiveanimals (29003, 29008, 29045, 29055, and 31042) 3 weeks later (59, 64). Three of these TVF-treated animals developed AIDS while on treatment;one animal, 29003, had TFV interrupted at the age of 95 weeks and developed AIDS at 177 weeks; animal 29045 was lost from the study at theage of 7 years due to an unrelated cause (self-injurious behavior). (B) Twelve newborn macaques were inoculated intravenously with a high doseof K65R mutants of SIV, and 3 weeks later, six of them were started on TFV as described previously (60). TFV-treated animal 29276 is currentlyAIDS free at the age of 12 years. (C) Four-week-old infant macaques were inoculated orally with SIVmac251, and three animals (32990, 33093,and 33109) were started on TFV treatment approximately 11 weeks later, when animals were already immunosuppressed (64). (D) Eleven juvenilemacaques (ages, 12 to 17 months) were inoculated orally with SIVmac251, and five animals were started on TFV treatment 2 weeks later, asdescribed previously (66). Two animals (32137 and 32993) developed AIDS, while the other three animals (32186, 33088, and 33091) are currentlyAIDS free after more than 6 years of SIV infection.



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from 45 to 319 fmol per million PBMC (Table 3). Four juvenilemacaques that were given a single subcutaneous dose of TFV(4 mg/kg) had TFV-DP concentrations of 24 to 76 fmol permillion PBMC 24 h after dosing. The average ratio of theintracellular TFV-DP concentration at 24 h postdose tothe mean extracellular TFV concentration in plasma over the24-h period was 0.47 for the chronically dosed animals,which was higher than the ratio of 0.27 for the single-dosedanimals (P � 0.027 by a two-tailed t test on log-transformedratios) (Table 3). This significant difference is indicative ofthe effective intracellular accumulation of TFV-DP overmultiple doses of TFV.

DISCUSSION

Data on the long-term safety and efficacy of TFV are im-portant, considering (i) the increasing use of TDF in regimensto treat HIV-infected persons, including in developing coun-tries, (ii) the ongoing preexposure prophylaxis trials investigat-ing whether daily administration of TDF can reduce the rate ofHIV infection among high-risk populations (1), and (iii) theanticipated regulatory approval of TDF for the treatment ofhepatitis B virus-infected persons. Because these groups in-clude women of reproductive age, available data on any long-term effects of in utero exposure to TFV are also very useful.While only long-term studies of humans can provide definitiveanswers, safety and efficacy data obtained from nonhumanprimate models of HIV infection are highly relevant, due tothe many similarities in physiology (including drug metabolism,infant development, kidney and bone physiology) and diseasepathogenesis (28, 36, 52, 66, 72). An advantage of the macaquemodel is that the relative durability of TFV’s efficacy and safetywithin a species can be evaluated in a more timely manner,because (i) without treatment, the disease course in macaques

TABLE 2. SIV-specific cell-mediated immune responses in long-term TFV-treated animals with low or undetectable levels of viremiaa

Animalno.

SIVstimulusb

CD4� CD3� T lymphocytes CD8� CD3� T lymphocytes

As % ofgated

lymphocytes

% Positive for:

As % ofgated

lymphocytes

% Positive for:

IL-2 IFN- TNF-IFN-�

andTNF-

IL-2 IFN- TNF- CD107a/b IFN-�andCD107a/b

TNF- andCD107a/b(% IFN-

�c)

IFN- andTNF- (%CD107a/

b�d)

29276 p27 37.2 0.014 0.103 0.177 0.110 23.4 0.014 0.350 0.764 0.510 0.140 0.630 (12.2) 0.110 (100.0)AT-2 36.6 0.014 0.103 0.197 0.110 23.2 0.028 0.000 0.000 0.030 0.000 0.000 (0.000) 0.000 (0.000)

30577 p27 48.6 0.002 0.073 0.177 0.066 14.1 0.025 1.333 1.860 2.080 1.400 1.570 (92.9) 1.210 (94.7)AT-2 49.0 0.003 0.233 0.397 0.230 14.5 0.000 0.113 0.420 0.000 0.130 0.310 (45.4) 0.140 (92.3)

33088 p27 38.7 0.000 0.008 0.012 0.000 27.2 0.011 0.000 0.000 0.000 0.000 0.000 (0.000) 0.000 (0.000)AT-2 37.8 0.002 0.130 0.273 0.150 26.5 0.008 0.000 0.000 0.030 0.000 0.000 (0.000) 0.000 (0.000)

33091 p27 32.1 0.008 0.217 0.400 0.210 15.9 0.028 0.100 0.160 0.170 0.000 0.000 (0.000) 0.000 (0.000)AT-2 31.7 0.008 0.307 0.610 0.310 15.6 0.005 0.000 0.070 0.220 0.000 0.000 (0.000) 0.000 (0.000)

32186 p27 54.8 0.000 0.023 0.077 0.042 29.0 0.000 0.039 0.040 0.000 0.000 0.000 (0.000) 0.000 (0.000)AT-2 55.0 0.000 0.121 0.283 0.140 29.1 0.027 0.004 0.015 0.000 0.000 0.000 (0.000) 0.000 (0.000)

a PBMC were collected after prolonged TFV treatment (12 years for animal 29276, 8 years for animal 30577, and 6 years for animals 32186, 33088, and 33091) andtested using flow cytometry assays with intracellular cytokine staining. Values are given after subtraction of the readings for medium-only control wells; negative valueswere assigned an arbitrary value of zero. Boldfaced data indicate positive immune responses (i.e., �2-fold above medium-only levels). Staphylococcus enterotoxin B wasused as a positive control to ensure cell viability and the ability of cells to secrete cytokines (data not shown).

b A peptide pool of SIVmac239gag p27 was used at 5 �g/ml; AT-2-inactivated SIVmac239 was used at 300 ng/ml.c Frequency of IFN--secreting cells as a percentage of the gated TNF-� CD107a/b� CD8� CD3� lymphocyte population.d Frequency of CD107a/b� cells as a percentage of the gated IFN-� TNF-� CD8� CD3� lymphocyte population.

TABLE 3. Intracellular levels of TFV-DP during single-dose orprolonged TFV treatmenta

Dose andanimal no.

TFVdose

(mg/kg)

Plasma TFVAUC0–24

(�g � h/ml)

IntracellularTFV-DP

concn(fmol/106

PBMC)24 h after

dosing

Ratio of�intracellularTFV-DP� at

24 h to�meanplasma

TFV� duringdosing

intervalb

Chronic dose29046 0.6 4.5 52 0.4029276 0.4 13.5 107 0.5730577 1.0 4.3 48 0.3831122 2.8 6.0 75 0.4332186 0.4 4.5 53 0.4133088 9.1 15.3 319 0.7233091 2.1 4.4 45 0.35

Single dose35391 4 6.0 76 0.4435410 4 4.1 29 0.3235434 4 5.8 31 0.1835942 4 4.2 24 0.19

a TFV was administered subcutaneously. Chronically dosed animals were onstable TFV regimens (administered once daily) for at least 7 weeks when the24-h pharmacokinetics study was performed (see Table 1); an exception wasanimal 29276, which was dosed three times per week. Blood collected at specifictime points after dosing was also used to isolate PBMC in order to measureintracellular TFV-DP levels. AUC0–24, AUC from 0 to 24 h.

b The ratio of the intracellular TFV-DP concentration (24 h after dosing) tothe mean plasma TFV concentration was calculated by assuming an approximatecell volume of 0.2 pl per cell (e.g., 100 fmol/106 PBMC means 500 nM); the meanplasma TFV concentration was calculated by dividing the AUC by the doseinterval and converting it also to nanomolar concentrations in order to calculatethe unitless ratio. The geometric mean of the ratios for chronically dosed animals(0.47) was significantly higher than that for single-dosed animals (0.27) (P �0.027 by a two-tailed t test on log-transformed ratios).



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infected with virulent SIV isolates is accelerated compared tothat in HIV-infected humans (41), and (ii) the whole develop-ment from infancy to adulthood and parenthood takes place inapproximately 4 to 6 years. So far, studies in macaque modelshave been quite predictive of the effects of TFV in humans andhave played an important role in guiding its clinical develop-ment. The data in the current report further support the fea-sibility of prolonged TFV treatment regimens.

All animals described in this report have in common thatthey have been on prolonged TFV regimens (administeredonce daily subcutaneously) for at least 1 year. At the initiationof these studies (from 1995 onward, i.e., prior to the clinicaldevelopment of the oral TDF regimen for humans), our un-derstanding of the interactions between pharmacologic, viro-logic, and host factors during TFV therapy was very limited.Accordingly, relatively high-dose subcutaneous TFV regimenswere used in an attempt to maximize antiviral efficacy. Follow-ing observations of toxicity, the dosage regimens were adjustedempirically to define the optimal window of drug exposure thatwould balance a minimal risk for toxicity with sufficient anti-viral activity (with the emergence of K65R viral mutants as aconfounding variable) to maintain overall health and delay theprogression of SIV disease. Over time, the ongoing collectionof safety and efficacy data from multiple macaque studies andthe ability to measure intracellular TFV-DP concentrationshave gradually refined our knowledge.

In the current follow-up studies, the primary dose-depen-dent toxicity continued to be renal toxicity, namely, the devel-opment of PRTD, in which chronic hypophosphatemia cansecondarily lead to bone disease, as described previously (63).No novel toxicities were observed in any other organ systems.Although the relatively small number of animals limits theability to detect small differences, serum parameters such asfasting concentrations of glucose, triglycerides, and cholesterolwere indistinguishable from those of age-matched untreatedanimals; this is consistent with the observations of favorablelipid profiles in HIV-infected people treated with TDF-con-taining regimens (24, 45).

The renal toxicity of high-dose TFV regimens in macaques isrelated to the renal drug clearance, which occurs through acombination of glomerular filtration and active tubular secre-tion (14, 16, 18). The degree of toxicity in tubular cells is likelydetermined by the local, intracellular concentration of TFV (orits phosphorylated metabolites), which is determined by thebalance between drug uptake from the plasma at the basolat-eral membrane (mediated by organic anion transporters) andthe active secretion of TFV at the apical brush-border mem-brane into the tubular lumen (mediated by ATP-binding cas-sette transporters such as MRP4) (14, 34, 51).

The current report provides additional insights into the cor-relation between age-dependent changes in pharmacokineticsand the likelihood of the development and resolution ofPRTD. As reported previously (63), no PRTD was detectedwith short-term high-dose TFV regimens in infant macaques,despite relatively high plasma drug concentrations (AUCs,�45 �g � h/ml). This is likely because the organic anion trans-porter mechanisms in the proximal renal tubuli are poorlydeveloped at birth (11, 39); thus, even with high plasma TFVconcentrations, the low uptake of TFV from the plasma by therenal tubular cells prevented accumulation to toxic intracellu-

lar concentrations. When infant macaques mature into juvenileanimals, these uptake mechanisms develop and result in ahigher renal clearance of TFV. However, the available datasuggest that for juvenile and adult macaques, if plasma TFVconcentrations are persistently high (AUCs, �20 �g � h/ml),the uptake of the drug from plasma (at the basolateral mem-brane) exceeds the secretion into the tubular lumen (at theapical brush-border membrane); in other words, the secretorytransport mechanisms at the apical membrane are the rate-limiting step in the renal secretion of TFV. In this situation, thegradual accumulation of TFV in these renal tubular cells leads,perhaps once a threshold is reached, to a dysfunction of thetubular cells that severely limits the transport of molecules inboth directions, resulting in significantly reduced TFV secre-tion (TFV clearance, �400 ml/h/kg), which coincides with thedetection of glucosuria and hypophosphatemia due to reducedreabsorption from the urinary filtrate. Once this process ofrenal toxicity is triggered, a vicious cycle is initiated in which,unless dosage reductions are implemented, increased exposureto TFV promotes further renal toxicity, the loss of proximaltubular cells, and eventually tubular atrophy (63).

In our studies with juvenile and adult macaques, our initialattempts to define a target window for plasma drug exposurewere complicated because a normal, physiologic decrease inrenal TFV clearance occurs when juvenile animals mature intoadulthood (Fig. 1). This is probably because for adult ma-caques, the kidney volume is independent of the animal’s sex,total body size, and weight (32, 33). Accordingly, for a TFVdosage regimen that is based on body weight (as is commonpractice in veterinary medicine), a heavy (due to muscle, fat, orpregnancy) adult macaque will experience higher plasma TFVAUCs than a lighter adult animal with kidneys of a similar size.In other words, as animals mature into adulthood, an increasein AUCs (and in the likelihood of PRTD development) can beavoided only by reducing the dosage regimen per kilogram ofbody weight, or dosing with a constant absolute amount of drug(irrespective of body weight fluctuations, but more reflective ofthe kidney capacity). The use of a constant dose is similar toroutine practice with most drugs for human adults, includingTDF, for which a fixed dose (one 300-mg tablet per day) isused within a relatively wide range of body weights (38).

The current data on several of the animals demonstratedthat during prolonged subcutaneous monotherapy with TFV,plasma concentrations lower (AUC, 4 to 10 �g � h/ml) thanour previously set target levels (AUC, 10 to 20 �g � h/ml [63])still effectively suppress virus replication. Such plasma AUCsare within a range two- to threefold higher than the steady-state AUCs (�3 �g � h/ml) obtained with the clinical dose ofTDF (one 300-mg tablet per day) for humans (4). In addition,intracellular TFV-DP concentrations in animals with plasmaAUCs of 4 to 15 �g � h/ml were 45 to 319 fmol per millionPBMC, values similar to those seen in humans chronicallytreated with oral TDF (�50 to 300 fmol/million PBMC [29,50]). Such similarities in drug concentrations underscore thegrowing relevance of these chronic low-dose TFV studies ofmacaques to model prolonged treatment regimens of humanswith TDF. The fact that the relatively low plasma TFV con-centrations in these chronically treated macaques providedintracellular concentrations that were sufficient to control vire-mia can be explained by the long intracellular half-life of



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TFV-DP in humans (median, 150 h; range, 60 to �175 h [29]),which is predicted to lead to intracellular accumulation. Con-sistent with this hypothesis, an increase of approximately two-fold in intracellular TFV-DP concentrations was observed fol-lowing multiple doses (Table 3). These pharmacokinetic dataalso suggest that in future macaque studies with prolongedsubcutaneous TFV regimens, the most rapid antiviral responsecan probably be achieved by a relatively high induction dose(aimed at quickly inducing sufficient intracellular TFV-DPconcentrations), followed by a lower-dose maintenance regi-men to avoid toxicity.

Although prolonged TFV monotherapy led to the selectionof K65R viral mutants in all animals, the virological responseswere variable, and this variability could not be explained bydrug concentrations or the presence of other RT mutations.Following the emergence of K65R mutants, some TFV-treatedanimals maintained persistent viremia and, although moreslowly than untreated animals, eventually progressed to AIDS.However, six TFV-treated animals were able to suppress K65Rviremia to undetectable or very low levels and to maintain thissuppression throughout the observation period (presently aslong as 12 years of infection). Although several of these ani-mals had PRTD (due to early high-dose regimens), their pro-longed AIDS-free survival is remarkable and, to our knowl-edge, unprecedented considering that (i) these animals wereinoculated with virus stocks that were very virulent (i.e., alluntreated animals had persistent viremia and developed dis-ease within several months or years), (ii) TFV monotherapywas used, and (iii) we and others have demonstrated that in theabsence of TFV treatment, K65R viral mutants are as virulentas wild-type virus (42, 60, 61). By either withdrawing TFVtreatment or depleting CD8� cells, we demonstrated previ-ously that both continued TFV therapy and CD8� cell-medi-ated immune responses were required to maximally suppressK65R viremia in such animals (58, 65). Accordingly, a combi-nation of factors (including antiviral immune responses anddrug-mediated effects) is responsible for the successful andsustained suppression of viremia in TFV-treated animals (58,65, 66). In other words, the variable virologic outcome amonganimals with K65R viral mutants but similar drug exposurelikely reflects the individual variability in antiviral immuneresponses that are needed to suppress these K65R mutants.While the TFV-treated animals with low or undetectable vire-mia showed measurable SIV-specific cell-mediated immuneresponses in peripheral blood, more research is needed tofurther elucidate the exact nature of these immune responses(e.g., epitope recognition, effector frequency in lymphoid tis-sues and at mucosal sites); information from such studies maybe useful for the development of better immunotherapeuticstrategies that can complement drug therapy. These findings ofreduced viremia of K65R SIV mutants associated with strongantiviral immune responses in TFV-treated macaques may ex-plain observations that viremia in persons with detectableK65R HIV-1 mutants can be suppressed by TFV-containingdrug regimens (13) and are consistent with clinical observa-tions of strong antiviral immune responses in HIV-1-infectedpeople receiving highly active antiretroviral therapy who havelow-level viremia with drug-resistant virus (2, 19).

An important concern about long-term HIV therapy is thesafety of a drug regimen during pregnancy (9). Growth restric-

tion had previously been reported for some newborn macaquesborn to adult female macaques treated during pregnancy witha high dose of TFV (30 mg/kg subcutaneously) (55, 56). How-ever, as explained previously (63), this was probably becausethis high-dose TFV regimen induced PRTD with phosphatedepletion in the mothers (and the reduced transplacentaltransfer of phosphate led to fetal deprivation), rather thanhaving a direct transplacental effect on the growing fetus. Inour studies, female macaque 31122 had been started at birthon continuous low-dose TFV treatment; although her plasmaTFV AUCs were four- to sixfold higher than those with theoral TDF regimen in humans, this animal has not demon-strated any signs of toxicity by the age of 10 years and hasdelivered three healthy offspring. These offspring showed nor-mal pre- and postnatal development (including renal parame-ters and bone development) throughout their observation pe-riods (as long as 5 years after birth). Although the number ofthese animals is small and therefore further investigation iswarranted, these preliminary observations with TFV duringpregnancy are consistent with the available data from the An-tiretroviral Pregnancy Registry, which show no indications ofan increased risk of birth defects after in utero exposure toTFV (3).

In conclusion, the current follow-up studies provide furtherinformation on the pharmacokinetics, safety, and efficacy oflong-term TFV regimens in macaques. While high-dose sub-cutaneous TFV regimens in macaques caused PRTD (whichwas managed with dosage reduction and, if needed, dietarysupplements), prolonged treatment with reduced doses thatmaintained plasma exposure levels below a certain thresholdwere safe and were associated with persistent virologic, immu-nologic, and clinical benefits in multiple animals. These obser-vations support the long-term treatment of HIV-infected hu-mans with TFV-containing regimens. Continued monitoring ofthese animals as they progress toward geriatric age will providefurther valuable information on prolonged treatment withTFV-containing regimens, with the ultimate goal of givingHIV-infected persons a normal life span.

ACKNOWLEDGMENTS

We thank P. Allen, V. Bakula, I. Cazares-Shaw, T. Dearman, L.Hirst, A. Ignatov, B. Rodello, W. von Morgenland, and the staff of theClinical Laboratories and Veterinary and Colony Services of the Cal-ifornia National Primate Research Center for expert technical assis-tance. The SIVmac239gag p27 peptides were obtained through theAIDS Research and Reference Reagent Program, Division of AIDS,NIAID, NIH. AT-2-inactivated SIVmac239 was provided by J. Lifson(SAIC Frederick, Inc., National Cancer Institute, Frederick, MD).

This research was supported by Gilead Sciences; E. Glaser PediatricAIDS Foundation grants PG-50609, PG-50757, and PG-50853 toK.K.A.V.R.; and grant RR-00169 from the National Center for Re-search Resources (NCRR; a component of NIH) to the CaliforniaNational Primate Research Center.

The contents of this article are solely the responsibility of the au-thors and do not necessarily represent the official views of the NCRRor NIH.

REFERENCES

1. AIDS Vaccine Advocacy Coalition. March 2005, posting date. Will a pill a dayprevent HIV? Anticipating the results of the tenofovir “PREP” trials. http://avac.org/pdf/tenofovir.pdf.

2. Alatrakchi, N., C. Duvivier, D. Costagliola, A. Samri, A. Marcelin, G.Kamkamidze, M. Astriti, R. Agher, V. Calvez, B. Autran, and C. Katlama.2005. Persistent low viral load on antiretroviral therapy is associated with Tcell-mediated control of HIV replication. AIDS 19:25–33.



http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

3. Antiretroviral Pregnancy Registry Steering Committee. 2007. AntiretroviralPregnancy Registry interim report for 1 January 1989 through 31 July 2007.Registry Coordinating Center, Wilmington, NC.

4. Barditch-Crovo, P., S. G. Deeks, A. Collier, S. Safrin, D. F. Coakley, M.Miller, B. P. Kearney, R. L. Coleman, P. D. Lamy, J. O. Kahn, I. McGowan,and P. S. Lietman. 2001. Phase I/II trial of the pharmacokinetics, safety, andantiretroviral activity of tenofovir disoproxil fumarate in human immunode-ficiency virus-infected adults. Antimicrob. Agents Chemother. 45:2733–2739.

5. Bedard, J., S. May, M. Lis, L. Tryphonas, J. Drach, J. Huffman, R. Sidwell,L. Chan, T. Bowlin, and R. Rando. 1999. Comparative study of the anti-human cytomegalovirus activities and toxicities of a tetrahydrofuran phos-phonate analogue of guanosine and cidofovir. Antimicrob. Agents Che-mother. 43:557–567.

6. Benech, H., F. Theodoro, A. Herbet, N. Page, D. Schlemmer, A. Pruvost, J.Grassi, and J. R. Deverre. 2004. Peripheral blood mononuclear cell countingusing a DNA-detection-based method. Anal. Biochem. 330:172–174.

7. Betts, M. R., and R. A. Koup. 2004. Detection of T-cell degranulation:CD107a and b. Methods Cell Biol. 75:497–512.

8. Blaas, S., A. Schneidewind, T. Gluck, and B. Salzberger. 2006. Acute renalfailure in HIV patients with liver cirrhosis receiving tenofovir: a report of twocases. AIDS 20:1786–1787.

9. Brogly, S. B., N. Ylitalo, L. M. Mofenson, J. Oleske, R. Van Dyke, M. J.Crain, M. J. Abzug, M. Brady, P. Jean-Philippe, M. D. Hughes, and G. R.Seage III. 2007. In utero nucleoside reverse transcriptase inhibitor exposureand signs of possible mitochondrial dysfunction in HIV-uninfected children.AIDS 21:929–938.

10. Buxbaum, S., F. B. Kraus, A. Hahn, O. Beck, B. Kabartas, H. W. Doerr, andB. Ludwig. 2006. Flow cytometric analysis of virus-specific T lymphocytes:practicability of detection of HCMV-specific T lymphocytes in whole bloodin patients after stem cell transplantation. J. Immunol. Methods 311:164–173.

11. Calcagno, P. L., and M. I. Rubin. 1963. Renal extraction of para-aminohip-purate in infants and children. J. Clin. Investig. 42:1632–1639.

12. Cassetti, I., J. V. Madruga, J. M. Suleiman, A. Etzel, L. Zhong, A. K. Cheng,and J. Enejosa. 2007. The safety and efficacy of tenofovir DF in combinationwith lamivudine and efavirenz through 6 years in antiretroviral-naive HIV-1-infected patients. HIV Clin. Trials 8:164–172.

13. Chappell, B. J., N. A. Margot, and M. D. Miller. 2007. Long-term follow-upof patients taking tenofovir DF with low-level HIV-1 viremia and the K65Rsubstitution in HIV-1 RT. AIDS 21:761–763.

14. Cihlar, T., E. S. Ho, D. C. Lin, and A. S. Mulato. 2001. Human renal organicanion transporter 1 (hOAT1) and its role in the nephrotoxicity of antiviralnucleotide analogs. Nucleosides Nucleotides Nucleic Acids 20:641–648.

15. Cundy, K. 1999. Clinical pharmacokinetics of the antiviral nucleotide ana-logues cidofovir and adefovir. Clin. Pharmacokinet. 36:127–143.

16. Cundy, K. C., C. Sueoka, G. R. Lynch, L. Griffin, W. A. Lee, and J.-P. Shaw.1998. Pharmacokinetics and bioavailability of the anti-human immunodefi-ciency virus nucleotide analog 9-[(R)-2-(phosphonomethoxy)propyl]adenine(PMPA) in dogs. Antimicrob. Agents Chemother. 42:687–690.

17. Davies, B., and T. Morris. 1993. Physiological parameters in laboratoryanimals and humans. Pharm. Res. 10:1093–1095.

18. Deeks, S. G., P. Barditch-Crovo, P. S. Lietman, F. Hwang, K. C. Cundy, J. F.Rooney, N. S. Hellmann, S. Safrin, and J. Kahn. 1998. Safety, pharmacoki-netics, and antiretroviral activity of intravenous 9-[2-(R)-(phosphonome-thoxy)propyl]adenine, a novel anti-human immunodeficiency virus (HIV)therapy, in HIV-infected adults. Antimicrob. Agents Chemother. 42:2380–2384.

19. Deeks, S. G., J. N. Martin, E. Sinclair, J. Harris, T. B. Neilands, H. T.Maecker, E. Hagos, T. Wrin, C. J. Petropoulos, B. Bredt, and J. M. McCune.2004. Strong cell-mediated immune responses are associated with the main-tenance of low-level viremia in antiretroviral-treated individuals with drug-resistant human immunodeficiency virus type 1. J. Infect. Dis. 189:312–321.

20. Durand-Gasselin, L., D. Da Silva, H. Benech, A. Pruvost, and J. Grassi.2007. Evidence and possible consequences of the phosphorylation of nucle-oside reverse transcriptase inhibitors in human red blood cells. Antimicrob.Agents Chemother. 51:2105–2111.

21. El Sahly, H. M., L. Teeter, T. Zerai, R. A. Andrade, C. Munoz, C. Nnabuife,and R. Hunter. 2006. Serum creatinine changes in HIV-seropositive patientsreceiving tenofovir. AIDS 20:786–787.

22. Fux, C. A., A. Christen, S. Zgraggen, M. G. Mohaupt, and H. Furrer. 2007.Effect of tenofovir on renal glomerular and tubular function. AIDS 21:1483–1485.

23. Gafni, R. I., R. Hazra, J. C. Reynolds, F. Maldarelli, A. N. Tullio, E. DeCarlo,C. J. Worrell, J. F. Flaherty, K. Yale, B. P. Kearney, and S. L. Zeichner. 2006.Tenofovir disoproxil fumarate and an optimized background regimen ofantiretroviral agents as salvage therapy: impact on bone mineral density inHIV-infected children. Pediatrics 118:e711–e718.

24. Gallant, J. E., S. Staszewski, A. L. Pozniak, E. DeJesus, J. M. Suleiman,M. D. Miller, D. F. Coakley, B. Lu, J. J. Toole, and A. K. Cheng. 2004.Efficacy and safety of tenofovir DF vs stavudine in combination therapy inantiretroviral-naive patients: a 3-year randomized trial. JAMA 292:191–201.

25. Gallant, J. E., M. A. Parish, J. C. Keruly, and R. D. Moore. 2005. Changes

in renal function associated with tenofovir disoproxil fumarate treatment,compared with nucleoside reverse-transcriptase inhibitor treatment. Clin.Infect. Dis. 40:1194–1198.

26. Giacomet, V., S. Mora, L. Martelli, M. Merlo, M. Sciannamblo, and A.Vigano. 2005. A 12-month treatment with tenofovir does not impair bonemineral accrual in HIV-infected children. J. Acquir. Immune Defic. Syndr.40:448–450.

27. Gitman, M. D., D. Hirschwerk, C. H. Baskin, and P. C. Singhal. 2007.Tenofovir-induced kidney injury. Expert Opin. Drug Saf. 6:155–164.

28. Gribnau, A. A. M., and L. G. M. Geijsberts. 1981. Developmental stages inthe rhesus monkey (Macaca mulatta). Springer-Verlag, Berlin, Germany.

29. Hawkins, T., W. Veikley, R. L. St Claire III, B. Guyer, N. Clark, and B. P.Kearney. 2005. Intracellular pharmacokinetics of tenofovir diphosphate, car-bovir triphosphate, and lamivudine triphosphate in patients receiving triple-nucleoside regimens. J. Acquir. Immune Defic. Syndr. 39:406–411.

30. Hazra, R., R. I. Gafni, F. Maldarelli, F. M. Balis, A. N. Tullio, E. DeCarlo,C. J. Worrell, S. M. Steinberg, J. Flaherty, K. Yale, B. P. Kearney, and S. L.Zeichner. 2005. Tenofovir disoproxil fumarate and an optimized backgroundregimen of antiretroviral agents as salvage therapy for pediatric HIV infec-tion. Pediatrics 116:e846–e854.

31. Hendrie, T. A., P. E. Peterson, J. J. Short, A. F. Tarantal, E. Rothgam, M. I.Hendrie, and A. G. Hendrickx. 1996. Frequency of prenatal loss in a ma-caque breeding colony. Am. J. Primatol. 40:41–53.

32. Hill, L. R., K. R. Hess, L. C. Stephens, P. T. Tinkey, and R. E. Price. 1999.Comparison of kidney weight and volume to selected anatomical parametersin the adult female rhesus monkey (Macaca mulatta). J. Med. Primatol.28:67–72.

33. Hill, L. R., K. R. Hess, L. C. Stephens, R. E. Price, and K. N. Gray. 2001.Correlation of kidney weight and volume and selected skeletal parametersto sex in the adult rhesus monkey (Macaca mulatta). J. Med. Primatol. 30:56–60.

34. Imaoka, T., H. Kusuhara, M. Adachi, J. D. Schuetz, K. Takeuchi, and Y.Sugiyama. 2007. Functional involvement of multidrug resistance-associatedprotein 4 (MRP4/ABCC4) in the renal elimination of the antiviral drugsadefovir and tenofovir. Mol. Pharmacol. 71:619–627.

35. Izzedine, H., J. S. Hulot, D. Vittecoq, J. E. Gallant, S. Staszewski, V. Launay-Vacher, A. Cheng, and G. Deray. 2005. Long-term renal safety of tenofovirdisoproxil fumarate in antiretroviral-naive HIV-1-infected patients. Datafrom a double-blind randomized active-controlled multicentre study. Neph-rol. Dial. Transplant. 20:743–746.

36. Jerome, C. P., and P. E. Peterson. 2001. Nonhuman primate models inskeletal research. Bone 29:1–6.

37. Jones, R., J. Stebbing, M. Nelson, G. Moyle, M. Bower, S. Mandalia, and B.Gazzard. 2004. Renal dysfunction with tenofovir disoproxil fumarate-con-taining highly active antiretroviral therapy regimens is not observed morefrequently. A cohort and case-control study. J. Acquir. Immune Defic. Syndr.37:1489–1495.

38. Kearney, B. P., J. F. Flaherty, and J. Shah. 2004. Tenofovir disoproxilfumarate: clinical pharmacology and pharmacokinetics. Clin. Pharmacoki-net. 43:595–612.

39. Lopez-Anaya, A., J. D. Unadkat, L. A. Schumann, and A. L. Smith. 1990.Pharmacokinetics of zidovudine (azidothymidine). II. Development of met-abolic and renal clearance pathways in the neonate. J. Acquir. ImmuneDefic. Syndr. 3:1052–1058.

40. Mauss, S., F. Berger, and G. Schmutz. 2005. Antiretroviral therapy withtenofovir is associated with mild renal dysfunction. AIDS 19:93–95.

41. McChesney, M. B., E. T. Sawai, and C. J. Miller. 1998. Simian immunode-ficiency virus, p. 322–345. In R. Ahmed and I. Chen (ed.), Persistent viralinfections. John Wiley & Sons, Ltd., New York, NY.

42. Metzner, K. J., J. M. Binley, A. Gettie, P. Marx, D. F. Nixon, and R. I.Connor. 2006. Tenofovir treatment augments anti-viral immunity againstdrug-resistant SIV challenge in chronically infected rhesus macaques. Ret-rovirology 3:97.

43. More, C., P. Bettembuk, H. P. Bhattoa, and A. Balogh. 2001. The effects ofpregnancy and lactation on bone mineral density. Osteoporos. Int. 12:732–737.

44. Moreno, S., P. Domingo, R. Palacios, J. Santos, V. Falco, J. Murillas, V.Estrada, J. Ena, and M. L. Alvarez. 2006. Renal safety of tenofovir disoproxilfumarate in HIV-1 treatment-experienced patients with adverse events re-lated to prior NRTI use: data from a prospective, observational, multicenterstudy. J. Acquir. Immune Defic. Syndr. 42:385–387.

45. Moyle, G. J., C. A. Sabin, J. Cartledge, M. Johnson, E. Wilkins, D. Churchill,P. Hay, A. Fakoya, M. Murphy, G. Scullard, C. Leen, and G. Reilly. 2006. Arandomized comparative trial of tenofovir DF or abacavir as replacement fora thymidine analogue in persons with lipoatrophy. AIDS 20:2043–2050.

46. National Research Council. 1996. Guide for the care and use of laboratoryanimals. National Academy Press, Washington, DC.

47. Nelson, M. R., C. Katlama, J. S. Montaner, D. A. Cooper, B. Gazzard, B.Clotet, A. Lazzarin, K. Schewe, J. Lange, C. Wyatt, S. Curtis, S. S. Chen, S.Smith, N. Bischofberger, and J. F. Rooney. 2007. The safety of tenofovirdisoproxil fumarate for the treatment of HIV infection in adults: the first 4years. AIDS 21:1273–1281.



http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

48. Padilla, S., F. Gutierrez, M. Masia, V. Canovas, and C. Orozco. 2005. Lowfrequency of renal function impairment during one-year of therapy withtenofovir-containing regimens in the real-world: a case-control study. AIDSPatient Care STDS 19:421–424.

49. Pozniak, A. L., J. E. Gallant, E. Dejesus, J. R. Arribas, B. Gazzard, R. E.Campo, S. S. Chen, D. McColl, J. Enejosa, J. J. Toole, and A. K. Cheng.2006. Tenofovir disoproxil fumarate, emtricitabine, and efavirenz versusfixed-dose zidovudine/lamivudine and efavirenz in antiretroviral-naive pa-tients: virologic, immunologic, and morphologic changes—a 96-week analy-sis. J. Acquir. Immune Defic. Syndr. 43:535–540.

50. Pruvost, A., E. Negredo, H. Benech, F. Theodoro, J. Puig, E. Grau, E. Garcia,J. Molto, J. Grassi, and B. Clotet. 2005. Measurement of intracellular di-danosine and tenofovir phosphorylated metabolites and possible interactionof the two drugs in human immunodeficiency virus-infected patients. Anti-microb. Agents Chemother. 49:1907–1914.

51. Ray, A. S., T. Cihlar, K. L. Robinson, L. Tong, J. E. Vela, M. D. Fuller, L. M.Wieman, E. J. Eisenberg, and G. R. Rhodes. 2006. Mechanism of active renaltubular efflux of tenofovir. Antimicrob. Agents Chemother. 50:3297–3304.

52. Roberts, J., E. W. Ford, and J. L. Southers. 1998. Urinary system, p. 312–323.In B. T. Bennett, C. R. Abee, and R. Henrickson (ed.), Nonhuman primatesin biomedical research diseases. Academic Press, San Diego, CA.

53. Sax, P. E., J. E. Gallant, and P. E. Klotman. 2007. Renal safety of tenofovirdisoproxil fumarate. AIDS Read. 17:90–92, 99–104, C3.

54. Shaw, J. P., C. M. Sueoka, R. Oliyai, W. A. Lee, M. N. Arimilli, C. Kim, andK. C. Cundy. 1997. Metabolism and pharmacokinetics of a novel oral pro-drug of 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) in dogs.Pharm. Res. 14:1824–1829.

55. Tarantal, A. F., M. L. Marthas, J.-P. Shaw, K. Cundy, and N. Bischofberger.1999. Administration of 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA)to gravid and infant rhesus macaques (Macaca mulatta): safety and efficacystudies. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 20:323–333.

56. Tarantal, A. F., A. Castillo, J. E. Ekert, N. Bischofberger, and R. B. Martin.2002. Fetal and maternal outcome after administration of tenofovir to gravidrhesus monkeys (Macaca mulatta). J. Acquir. Immune Defic. Syndr. 29:207–220.

57. Tsai, C.-C., K. E. Follis, T. W. Beck, A. Sabo, R. F. Grant, N. Bischofberger,and R. E. Benveniste. 1995. Prevention of simian immunodeficiency virusinfection in macaques by 9-(2-phosphonylmethoxypropyl)adenine (PMPA).Science 270:1197–1199.

58. Van Rompay, K. K., J. A. Johnson, E. J. Blackwood, R. P. Singh, J. Lips-comb, T. B. Matthews, M. L. Marthas, N. C. Pedersen, N. Bischofberger, W.Heneine, and T. W. North. 2007. Sequential emergence and clinical impli-cations of viral mutants with K70E and K65R mutation in reverse transcrip-tase during prolonged tenofovir monotherapy in rhesus macaques withchronic RT-SHIV infection. Retrovirology 4:25.

59. Van Rompay, K. K. A., J. M. Cherrington, M. L. Marthas, C. J. Berardi, A. S.Mulato, A. Spinner, R. P. Tarara, D. R. Canfield, S. Telm, N. Bischofberger,and N. C. Pedersen. 1996. 9-[2-(Phosphonomethoxy)propyl]adenine therapyof established simian immunodeficiency virus infection in infant rhesus ma-caques. Antimicrob. Agents Chemother. 40:2586–2591.

60. Van Rompay, K. K. A., J. M. Cherrington, M. L. Marthas, P. D. Lamy, P. J.Dailey, D. R. Canfield, R. P. Tarara, N. Bischofberger, and N. C. Pedersen.1999. 9-[2-(Phosphonomethoxy)propyl]adenine (PMPA) therapy prolongssurvival of infant macaques inoculated with simian immunodeficiency viruswith reduced susceptibility to PMPA. Antimicrob. Agents Chemother. 43:802–812.

61. Van Rompay, K. K. A., M. D. Miller, M. L. Marthas, N. A. Margot, P. J.Dailey, R. P. Tarara, D. R. Canfield, J. M. Cherrington, N. L. Aguirre, N.Bischofberger, and N. C. Pedersen. 2000. Prophylactic and therapeutic ben-efits of short-term 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA)administration to newborn macaques following oral inoculation with simianimmunodeficiency virus with reduced susceptibility to PMPA. J. Virol. 74:1767–1774.

62. Van Rompay, K. K. A., M. B. McChesney, N. L. Aguirre, K. A. Schmidt, N.Bischofberger, and M. L. Marthas. 2001. Two low doses of tenofovir protectnewborn macaques against oral simian immunodeficiency virus infection.J. Infect. Dis. 184:429–438.

63. Van Rompay, K. K. A., L. L. Brignolo, D. J. Meyer, C. Jerome, R. Tarara, A.Spinner, M. Hamilton, L. L. Hirst, D. R. Bennett, D. R. Canfield, T. G.Dearman, W. Von Morgenland, P. C. Allen, C. Valverde, A. B. Castillo, R. B.Martin, V. F. Samii, R. Bendele, J. Desjardins, M. L. Marthas, N. C.Pedersen, and N. Bischofberger. 2004. Biological effects of short-term andprolonged administration of 9-[2-(phosphonomethoxy)propyl]adenine(tenofovir) to newborn and infant rhesus macaques. Antimicrob. AgentsChemother. 48:1469–1487.

64. Van Rompay, K. K. A., R. Singh, L. Brignolo, J. R. Lawson, K. A. Schmidt,B. Pahar, D. R. Canfield, R. P. Tarara, N. Bischofberger, and M. Marthas.2004. The clinical benefits of tenofovir for simian immunodeficiency virus-infected macaques are larger than predicted by its effects on standard viraland immunological parameters. J. Acquir. Immune Defic. Syndr. 36:900–914.

65. Van Rompay, K. K. A., R. P. Singh, B. Pahar, D. L. Sodora, C. Wingfield,J. R. Lawson, M. L. Marthas, and N. Bischofberger. 2004. CD8� cell-mediated suppression of virulent simian immunodeficiency virus duringtenofovir treatment. J. Virol. 78:5324–5337.

66. Van Rompay, K. K. A. 2005. Antiretroviral drug studies in non-humanprimates: a valid animal model for innovative drug efficacy and pathogenesisstudies. AIDS Rev. 7:67–83.

67. Van Rompay, K. K. A., B. P. Kearney, J. J. Sexton, R. Colon, J. R.Lawson, E. J. Blackwood, W. A. Lee, N. Bischofberger, and M. L. Mar-thas. 2006. Evaluation of oral tenofovir disoproxil fumarate and topicaltenofovir GS-7340 to protect infant macaques against repeated oral chal-lenges with virulent simian immunodeficiency virus. J. Acquir. ImmuneDefic. Syndr. 43:6–14.

68. Vela, J. E., L. Y. Olson, A. Huang, A. Fridland, and A. S. Ray. 2007.Simultaneous quantitation of the nucleotide analog adefovir, its phosphory-lated anabolites and 2 -deoxyadenosine triphosphate by ion-pairing LC/MS/MS. J. Chromatogr. B 848:335–343.

69. Vigano, A., G. V. Zuccotti, L. Martelli, V. Giacomet, L. Cafarelli, S. Bor-gonovo, S. Beretta, G. Rombola, and S. Mora. 2007. Renal safety of tenofovirin HIV-infected children: a prospective, 96-week longitudinal study. Clin.Drug Investig. 27:573–581.

70. Winston, A., J. Amin, P. Mallon, D. Marriott, A. Carr, D. A. Cooper, and S.Emery. 2006. Minor changes in calculated creatinine clearance and anion-gap are associated with tenofovir disoproxil fumarate-containing highly ac-tive antiretroviral therapy. HIV Med. 7:105–111.

71. Zimmermann, A. E., T. Pizzoferrato, J. Bedford, A. Morris, R. Hoffman, andG. Braden. 2006. Tenofovir-associated acute and chronic kidney disease: acase of multiple drug interactions. Clin. Infect. Dis. 42:283–290.

72. Zoetis, T., and M. Hurtt. 2003. Species comparison of anatomical and func-tional renal development. Birth Defects Res. B 68:111–120.



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