Efficacy and Adverse Effects of the Antiviral Compound Plerixaforin Feline Immunodeficiency Virus-Infected Cats

K. Hartmann, C. Stengel, D. Klein, H. Egberink, and J. Balzarini

Background: Bicyclam derivatives inhibit feline immunodeficiency virus (FIV) replication through selective blockage of

chemokine receptor CXCR4.

Hypothesis/Objectives: CXCR4 antagonist plerixafor (AMD3100, 1,1′-bis-1,4,8,11-tetraazacyclotetradekan) alone or

combination with adefovir (PMEA, 9-(2-phosphonylmethoxyethyl)adenine) safe and effective for treating FIV-infected

cats.

Animals: Forty naturally FIV-infected, privately owned cats.

Materials and Methods: Prospective, placebo-controlled, double-blind clinical trial. Cats randomly classified into 4

treatment groups. Received AMD3100, PMEA, AMD3100 in combination with PMEA, or placebo for 6 weeks. Clinical

and laboratory parameters, including CD4+ and CD8+ cell counts, FIV proviral and viral load measured by quantitative

polymerase chain reaction (qPCR) evaluated. Additionally, FIV isolates from cats treated with AMD3100 tested for drug

resistance.

Results: FIV-infected cats treated with AMD3100 caused significant decrease in proviral load compared to placebo

group (2.3 ± 3.8% to 1.9 ± 3.1%, of blood lymphocytes P < .05), but did not lead to improvement of clinical or immuno-

logical variables; it caused a decrease in serum magnesium concentration without clinical signs. No development of resis-

tance of FIV isolates to AMD3100 found during treatment period. PMEA administration improved stomatitis (stomatitis

score [degree 1 – 100] PMEA group: 23 ± 19 to 11 ± 10, P < .001; AMD3100 + PMEA group: 12 ± 17 to 3 ± 5, P < .05),

but did not decrease proviral or viral load and caused anemia (RBC [9106/lL] PMEA group: 9.07 ± 1.60 to 6.22 ± 2.16,

P < .05; AMD3100 ± PMEA group: 8.80 ± 1.23 to 5.84 ± 1.58, P < .001).

Conclusions and Clinical Importance: Administration of CXCR4 antagonists, as AMD3100, can induce reduction of

proviral load and may represent viable treatment of FIV-infected cats. Combination treatment with PMEA not recom-

mended.

Key words: AMD3100; Bicyclam; CXCR4; FIV.

Feline immunodeficiency virus (FIV) was firstdetected in 1986,1 and is recognized as a common

infectious agent of domestic cats worldwide. Mode ofinfection, cell types infected, dispersal of virus in thebody, and time course of infection in cats closelyresemble those features in human immunodeficiencyvirus (HIV) infection in people.2 Both HIV and FIVuse a chemokine receptor for infecting cells.3,4 Chemo-kine receptors belong to the group of transmembraneproteins, in which signal transmission is affordedthrough rapid influx of calcium into the cell. They arealso essential co-receptors for HIV and FIV duringinfection of primary susceptible CD4+ lymphocytes.5

In late-stage HIV infection, viral isolates mainly usethe CXCR4 receptor for cell entry.6 In FIV infection,

CXCR4 also is one of the major co-receptors.4,7 Byblocking the chemokine receptors, infection of cells byHIV or FIV can be prevented.4,8,9

From the Clinic of Small Animal Medicine, LMU University ofMunich, Munich, Germany (Hartmann, Stengel); the VetcoreFacility for Research, University of Veterinary Medicine, Vienna,Austria (Klein); the Institute of Virology, Faculty of VeterinaryMedicine, Utrecht University, Utrecht, The Netherlands(Egberink); and the Laboratory of Virology and Chemotherapy,Rega Institute for Medical Research, Katholieke UniversiteitLeuven, Leuven, Belgium (Balzarini). Location: Research wasperformed at the Clinic of Small Animal Medicine, LMUUniversity of Munich, Munich, Germany.

Corresponding author: Katrin Hartmann, Clinic of Small Ani-mal Medicine, LMU University of Munich, Veterinaerstrasse 13,80539 Munich, Germany, e-mail: hartmann@lmu.de.

Submitted July 28, 2011; Revised January 4, 2012;Accepted January 27, 2012.

Copyright © 2012 by the American College of Veterinary InternalMedicine

10.1111/j.1939-1676.2012.00904.x

Abbreviations:

HIV human immunodeficiency virus

FIV feline immunodeficiency virus

AMD3100 plerixafor, 1,1′-[1,4-phenylenbismethylene]-bis

(1,4,8,11-tetraazacyclotetradecane)-octachloride

dihydrate

PMEA adefovir 9-(2-phosphonylmethoxyethyl)adenine

qPCR quantitative polymerase chain reaction

DNA deoxyribonucleic acid

RNA ribonucleic acid

dATP deoxyadenosine triphosphate

dCTP deoxycytidine triphosphate

dGTP deoxyguanosine triphosphate

dUTP deoxyuridine triphosphate

CrFK Crandell feline kidney cells

rDNA ribosomal deoxyribonucleic acid

RT reverse trancriptase

HAART highly active antiretroviral therapy

FeLV feline leukemia virus

PBS phosphate-buffered saline

RT reverse transcriptase

EC50 50% effective concentration

TCID50 50% tissue culture infective dose

RBC red blood cell count

NK natural killer cells

PMEApp diphosphorylated PMEA

AZT zidovudine, azidothymidine

PMPA tenofovir, 9-(2-phosphonylmethoxypropyl)adenine

J Vet Intern Med 2012;26:483–490

Bicyclams are low molecular weight nonpeptidiccompounds that bind selectively to the chemokinereceptor CXCR4,10 thereby preventing interaction ofthis receptor with other ligands, such as HIV or FIV,and inhibiting entry of these viruses into the cell.10–13

Plerixafor (1,1′-[1,4-phenylenbismethylene]-bis(1,4,8,11-tetraazacyclotetradecane)-octachloride dehydrate, AM-D3100, JM3100), is the prototype compound amongthe bicyclams. Plerixafor is commercially availablea

and used for stem cell mobilization in humans.14 In vi-tro studies showed that AMD3100 efficiently inhibitsFIV replication.12,15

Highly active antiretroviral treatment (HAART)protocols in which compounds of several classes arecombined are currently the mainstay of treatment ofHIV-infected people. This has led to a dramaticimprovement of prognosis and survival in infectedhumans.16 Although several anti-HIV compounds havebeen shown to inhibit FIV replication in cell culture,antiviral chemotherapy is not widely used inFIV-infected cats. Zidovudin (AZT, azidothymidine,3′-azido-2′, 3′-dideoxythymidine) is the only drug cur-rently applied to some naturally FIV-infected cats, butis not very effective and can be associated with severeadverse effects.17–19 Multidrug protocols have onlybeen used in case reports,20 and in vivo efficacy studiesin naturally FIV-infected cats are missing for mostHIV compounds, including chemokine receptor block-ers. All drugs so far investigated in controlled fieldstudies in cats are nucleoside analogs, and combinationwith other drug classes has not been investigated.

Therefore, the aim of the present study was toevaluate efficacy and adverse effects of the bicyclamplerixafor and the nucleoside analog adefovir (9-(2-phosphonylmethoxyethyl)adenine (PMEA),b alone orin combination in FIV-infected cats in a placebo-controlled, double-blind study specifically looking at(1) quality of life measures and clinical signs, (2)changes in CD4+ and CD8+ counts, (3) decrease inproviral and viral load, (4) adverse effects, and (5)development of drug resistance.

Materials and Methods

Study Design

The study was designed as a 6-week placebo-controlled,

double-blinded, clinical trial. It fulfilled the general German

guidelines for prospective studies with owners’ consent. Forty

cats were included and randomly assigned to 1 of 4 groups of 10

cats each. Ten cats received placebo only, 10 cats received

AMD3100 and placebo, 10 cats received PMEA and placebo,

and 10 cats received AMD3100 and PMEA.

Animals

Cats were only included in the study if they were FIV-infected

and had clinically evident stomatitis. All cats had serum antibodies

against FIV p24 antigen detected by ELISAc and detectable provi-

rus in blood measured by quantitative polymerase chain reaction

(qPCR). Only cats for which the owners gave their consent for par-

ticipation were included. Cats were excluded if they were coinfected

with feline leukemia virus (FeLV), if they were in a moribund con-

dition (Karnofsy’s score < 30%), or if they were aggressive.

Thirty-six of the 40 cats (90%) were domestic short-hair cats

and 4 (10%) were long-hair mixed-breed cats. Age of cats ranged

between 1 and 11 years (median 6.4 years). All animals were

neutered; 28/40 (70%) were male, 12/40 (30%) were female.

Drugs

AMD3100a and its respective placebo were injected SC at

0.5 mg/kg q12h; PMEAb and its respective placebo were injected

SC at 10 mg/kg twice weekly. Sterile phosphate-buffered saline

(PBS) was used as placebo and as solvent for PMEA and

AMD3100. Preparation and encoding of all compounds was per-

formed at the Rega Institute for Medical Research (JB). All other

investigators were masked until completion of statistical analysis

of all data.

Variables Investigated

To determine efficacy and detect adverse effects, proviral and

viral load, CD4+ and CD8+ counts, clinical signs, and laboratory

parameters were monitored throughout the study. A physical

examination was performed in all cats before treatment and then

weekly during the trial. A numerical scoring system (0 = no clinical

sign; 100 = most severe signs) was designed to semiquantify sever-

ity of stomatitis and conjunctivitis. To enable investigators to

assess the overall health of the animals objectively, the Karnofsky’s

score adapted to cats21 was used. To detect adverse effects, a com-

plete blood count (CBC)d and serum biochemistry analysise were

performed every second week. Proviral and viral load and CD4+

and CD8+ cell counts were measured every 2nd week as follows.

Lymphocytes were stained for cell surface expression of CD3,

CD4, and CD8 as described,22 and counted using a fluorescence-

activated cell sorter.f In general, 30,000 events were acquired and

analyzed using 2 different software programs.g CD4+ and CD8+

cell counts and the CD4 : CD8 ratio were calculated.

Proviral and viral load was determined by Taqman qPCR.

Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) were

extracted from 200 lL of whole blood or 140 lL of plasma,

respectively, using 2 extraction kits.h,i The FIV proviral load in

peripheral blood mononuclear cells (PBMC) was quantified using

qPCR measuring PCR product accumulation through a dual-

labeled fluorogenic TaqMan probe. Because a broad range of

FIV subtypes likely occur in the area where the study was

performed,23 3 different assays targeting a broad range of FIV

subtypes (1010p, 1372p, and 1416p) that have been described

previously,24–26 were used in the present study. The 25-lL PCR

mixture contained 10 mM Tris (pH 8.3), 50 mM KCl, 3 mM

MgCl2, 200 nM deoxyadenosine triphosphate (dATP), deoxycyti-

dine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP),

400 nM deoxuridine triphosphate (dUTP), 300 nM of each

primer, 200 nM of the fluorogenic probe, and 2.5 units of Taq

DNA polymerase. After initial denaturation (2 minutes at 95°C),amplification was performed with 45 cycles of 15 seconds at 95°Cand 60 seconds at 60°C. The PCR reaction and the on-line mea-

surement of the emitted fluorescence were performed on a

sequence detector system.j The copy number per PCR reaction

was calculated using sequence detection softwarek utilizing a

series of 4-fold dilutions of genomic DNA derived from a Crandell

feline kidney (CrFK) cell line infected with FIV (Petaluma). The

absolute DNA content per PCR reaction was estimated by a

second real-time PCR assay targeting the 18S ribosomal deoxyri-

bonucleic acid (rDNA) genes.26 The relative proviral load was

calculated by setting the value at beginning of therapy as 100%

and then relating all subsequent measurements to this.

484 Hartmann et al

The FIV viral load in plasma was quantified with reverse

transcriptase (RT) qPCR using the same primers and probes as

above. The 25-lL one-step RT qPCR mixture contained 12.5 lL29 reaction buffer,l 300 nM of each primer, 200 nM fluorogenic

probe, 0.5 lL SuperScript III RT/Platinium Taq Mix, and 5 lLof sample. After a reverse transcription step (30 minutes at 42°C)followed by a denaturation step (10 minutes at 95°C), amplifica-

tion was performed with 45 cycles of 15 seconds at 95°C and

60 seconds at 60°C. Reverse transcription and amplification were

performed in a sequence detection system.m The copy number

per RT qPCR reaction was calculated by sequence detection soft-

ware k utilizing a series of 10-fold dilutions of in vitro transcribed

RNA as described.25

Development of Resistance

A blood sample for virus isolation was analyzed in all cats

receiving AMD3100 and all cats receiving AMD3100 and PMEA

on days 0 and 42. Virus isolates were titrated and used in an

antiviral assay. Thus, 50% effective concentration (EC50) values

were determined by adding different concentrations of AMD3100

to thymocytes cultured in 96-well plates with 100 50% tissue cul-

ture infective doses (TCID50) of FIV per well. Virus production

was measured by p24 ELISA,27 and the highest concentrations of

AMD3100 inducing 50% p24 levels were estimated. The EC50

values on days 0 and 42 were compared.

Statistical Evaluation

Clinical stomatitis and conjunctivitis scores, modified Karnof-

sky’s index values, all laboratory parameters, lymphocyte subset

numbers, and proviral and viral load were compared among the

4 treatment groups by use of one-way ANOVA conducted using

SPSS.n Whenever a statistically significant difference was detected

among groups, the change over time was determined by calculat-

ing the difference between the mean values at the end and begin-

ning of therapy. The Kruskall-Wallis Test was then performed to

analyze differences among separate groups. To ensure that the 4

groups were comparable concerning the variables investigated

before treatment initiation, groups were compared on day 0 using

one-way ANOVA. A P value < .05 was considered significant for

all analyses.

Results

Efficacy of the Compounds

There was no significant difference in any evaluatedvariable among the 4 groups before treatment initia-tion on day 0 (Table 1). At the end of the study, allFIV-infected cats were still alive. During the treatmentperiod, the clinical status of all cats improved in allgroups. Among clinical variables, no significant differ-ence was seen in the body weight, the Karnofsky’sscore, or the conjunctivitis score among treatmentgroups. A statistically significant difference wasdetected only for the stomatitis score. Improvement instomatitis score for cats receiving PMEA only(P < .001) or PMEA with AMD3100 (P < .05) wassignificantly greater than for cats receiving placebo(Fig 1). By contrast, no significant difference inimprovement of stomatitis was detected between catsreceiving AMD3100 and cats receiving placebo.

No significant changes in absolute CD4+ or CD8+

counts or the CD4 : CD8 ratio were observed among

groups. There were also no significant differences inviral load among groups. However, significant differ-ences in proviral load were detected among groups;compared to the placebo group, the relative proviralload decreased significantly in the AMD3100 group(P < .05), but a relative increase was observed in catsreceiving PMEA and AMD3100 (P < .05) comparedto cats receiving only AMD3100 (Fig 2).

Adverse Effects

A significant decrease in red blood cell (RBC)counts (Fig 3), hemoglobin concentration, and hemat-ocrit was found in cats receiving PMEA in comparisonto cats receiving placebo (P < .05) and compared tocats receiving AMD3100 (P < .05), as well as in catsreceiving both compounds (PMEA + AMD3100) incomparison to the placebo group (P < .001) and incomparison to cats receiving AMD3100 (P < .001).No significant differences were found in the whiteblood cell counts. Among all biochemical parametersevaluated, only serum magnesium decreased signifi-cantly in cats receiving AMD3100 (P < .05) and incats receiving PMEA and AMD3100 (P < .05) com-pared to placebo-treated cats as well as in cats receiv-ing PMEA and AMD3100 compared to cats receivingPMEA only (P < .05) (Fig 4). Changes in concentra-tions of calcium and other electrolytes were not signifi-cantly different among groups.

Development of Resistance

In 15 cats receiving AMD3100 (7 cats) orAMD3100 and PMEA (8 cats), virus isolation waspossible on day 0 and day 42. The 50% effective con-centration (EC50) values against AMD3100 were deter-mined for 15 FIV isolates. For the FIV isolates thatcould be recovered from the AMD3100-treated cats,EC50 values before (day 0) and after (day 42) treat-ment were 52 ± 46 nM and 68 ± 89 nM, respectively.The EC50 values for the FIV isolates before and afterAMD3100 + PMEA treatment were 135 ± 64 nM and200 ± 150 nM, respectively. In both groups of virusisolates there was no statistically significant differencein the susceptibility to AMD3100 before or after drugtreatment revealing that no drug resistance develop-ment occurred within the treatment period.

Discussion

Both drugs used in this study showed evidence ofefficacy but also mild adverse effects. No significant dif-ference in overall health status was detected betweencats receiving AMD3100 and cats receiving placebo orPMEA. However, the stomatitis score improved in catsfrom the PMEA-treated and AMD3100/PMEA-treatedgroup. By contrast, stomatitis in cats treated solelywith AMD3100 did not improve. This might bebecause AMD3100 exerts an antiretroviral effectagainst FIV but is not active against other viruses, suchas feline herpesvirus28 or calicivirus considered impor-

AMD3100 Treatment against FIV 485

tant cofactors in the development of FIV-associatedstomatitis. Instead, as demonstrated in earlier studies,an improvement or cure of stomatitis in FIV-infectedcats was seen in treatment with PMEA or relateddrug.17,29 Nucleoside phosphonates such as PMEA areeffective in vivo against a broad spectrum of retrovirus-es, including HIV and FIV,30 and several DNA virusesincluding certain herpesviruses.29,31 Antiproliferativeeffects32 and immunomodulatory properties (eg,through intensification of natural killer cell [NK] activ-ity and interferon production)33,34 also occur duringPMEA treatment. Because herpesvirus and calicivirusare common causes of stomatitis in cats,35 PMEAmight have exerted a direct antiviral effect on theseviruses. Alternatively, it is possible that PMEA inhib-

ited excessive growth of the oral mucosa because of itsantiproliferative properties. This could explain theimprovement of stomatitis in cats of both groupsreceiving PMEA in contrast to the other groups. Itshould be noticed that cats treated with PMEA startedwith more severe average stomatitis scores (althoughthis difference was not statistically significantly differentbetween the groups; see Table 1) which markedlyresolved by day 42 and that it cannot be fully excludedthat improvement of stomatitis was not a direct effectof the drug but rather a coincidental cycling in thedegree of stomatitis as clinical signs may fluctuate withor without treatment.

There was a significantly reduced relative proviralload in AMD3100-treated cats in the present study.

Table 1. Values of the investigated variables in all 4 treatment groups before treatment initiation (day 0). Therewas no significant difference in any variable among the 4 groups when compared by one-way ANOVA (P > .05).

Variables Placebo AMD3100 PMEA AMD3100 + PMEA

Body weight (kg) Mean 5.3 5.0 5.0 4.7

SD 1.8 1.4 1.3 1.3

Range 2.6–7.5 2.6–7.1 3.3–6.7 2.9–6.2Karnofsky’s score (%) Mean 83.1 93.4 81.4 85.9

SD 17.5 4.1 13.3 16.8

Range 55.0–98.0 85.0–98.0 60.0–96.0 50.0–99.0Stomatitis score (degree 1–100) Mean 9.0 4.2 23.2 12.0

SD 12.5 9.1 18.6 16.8

Range 0.0–35.0 0.0–30.0 0.0–50.0 0.0–50.0Conjunctivitis score (degree 1–100) Mean 3.4 4.0 5.8 5.3

SD 6.4 6.7 9.3 7.8

Range 0.0–20.0 0.0–20.0 0.0–30.0 0.0–25.0CD4+ cell count (/lL) Mean 417 532 545 1,002

SD 334 776 349 1,655

Range 61–995 93–2,103 45–1,138 64–4,352CD8+ cell count (/lL) Mean 379 503 530 357

SD 428 594 309 310

Range 80–1,240 114–1,596 34–995 50–827CD4/CD8 ratio Mean 1.28 1.22 1.15 2.25

SD 0.65 0.78 0.50 1.86

Range 0.74–2.34 0.29–2.54 0.31–2.01 0.77–5.26Provirus load Mean 6.06 2.33 0.82 3.15

(% infected PBL) SD 18.17 3.81 1.76 6.86

Range 0.01–57.76 0.01–12.46 0.06–5.80 0.00–22.49Virus load (copy RNA/mL plasma) Mean 348,773 5,007 1,269 88,518

SD 1,093,000 8,040 748 251,088

Range 0–3,459,000 0–26,263 228–748 3–757,755RBC (9106/lL) Mean 8.57 8.79 9.07 8.80

SD 1.69 1.66 1.6 1.23

Range 4.65–10.20 6.26–11.30 6.55–10.80 5.69–10.00Hb (mmol/L) Mean 7.42 7.77 7.96 7.48

SD 1.60 1.20 1.40 0.90

Range 4.28–9.31 5.91–9.00 4.66–9.75 5.90–8.63PCV (%) Mean 36.6 38.2 39.5 36.4

SD 7.5 5.7 6.4 4.2

Range 21.0–45.0 29.0–45.0 24.0–48.0 29.0–41.0Mg++ (mmol/L) Mean 0.86 0.87 0.91 0.82

SD 0.10 0.08 0.09 0.10

Range 0.60–0.96 0.69–1.00 0.71–1.03 0.60–1.00Ca++ (mmol/L) Mean 2.48 2.50 2.44 2.49

SD 0.11 0.12 0.05 0.11

Range 2.40–2.70 2.30–2.70 2.40–2.50 2.30–2.70

SD, standard deviation; RBC, red blood cells; Hb, hemoglobin; PCV, packed cell volume; Mg++, magnesium; Ca++, calcium; PBL,

peripheral blood lymphocytes.

486 Hartmann et al

Although statistically significant, the effect was notvery pronounced; however, it demonstrates for the firsttime antiviral efficacy of this compound in naturallyFIV-infected cats. A more pronounced effect on theprovirus load could have potentially been achievedduring a longer treatment period. It is also possiblethat the virus load even further decreased more (andother parameters continued to improve) after treat-ment was stopped. This, however, was unfortunatelynot investigated in the study because cats were notavailable for further rechecks. The demonstrated anti-viral efficacy is supported by cell culture data revealingthat AMD3100 causes a dose-dependent inhibition of

FIV replication comparable to that seen againstHIV.10,12,36,37 In HIV-infected humans, efficacy wasshown in a Phase II clinical trial38 but not further pur-sued mainly attributable to lack of oral bioavailabilityand an unexpected stem cell mobilizing effect.14 How-ever, in contrast to results from the present study, datafrom an experimental study39 revealed that AMD3100failed to decrease proviral load when administered tochronically FIV-infected cats.39 However, in this previ-ous study,39 all cats were infected with one specificFIV isolate in an experimental setting, whereas in thepresent study cats were naturally infected and presum-ably with different FIV strains, which might explain thedifference in proviral load development. Indeed, it hasbeen reported that FIV can use alternative co-recep-tors.40 This could have influenced the outcome of theexperimental study if one assumes that the efficacy ofusing an alternative coreceptor can differ depending the

Fig 2. Changes in the relative FIV provirus load (in %) during

the 6-week treatment in cats receiving placebo, AMD3100,

PMEA, or AMD3100 and PMEA. The provirus load before initi-

ation of the treatment (day 0) was set to 100%, and the values

on day 14, day 28, and day 42 were shown as % of the value of

day 0. Bars demonstrate the change in the provirus load (in %)

on day 14, day 28, and day 42 of all cats in the respective

groups. Changes in proviral load were significantly different

between cats receiving placebo versus cats receiving AMD3100

(P < .05) and between cats receiving PMEA and AMD3100 com-

pared to cats receiving only AMD3100 (P < .05).

Fig 1. Changes in the stomatitis score (degree = 0–100; demon-

strated by a numerical scoring system: 0 = no clinical sign;

100 = most severe signs) during the 6-week treatment in cats

receiving placebo, AMD3100, PMEA, or AMD3100 and PMEA.

Bars demonstrate the degree of stomatitis on day 0, day 14, day

28, and day 42 of all cats in the respective groups. Changes in

the stomatitis score were significantly different between cats

receiving PMEA only compared to cats receiving placebo

(P < .001) and in cats receiving PMEA and AMD3100 compared

to cats receiving placebo (P < .05). There was no significant dif-

ference between cats receiving AMD3100 and cats receiving pla-

cebo.

Fig 4. Changes in serum magnesium (in mmol/L) during the

6-week treatment in cats receiving placebo, AMD3100, PMEA,

or AMD3100 and PMEA. Bars demonstrate the serum magne-

sium concentration on day 0, day 14, day 28, and day 42 of all

cats in the respective groups. There was a significant difference

between cats receiving AMD3100 versus cats receiving placebo

(P < .05) and in cats receiving PMEA and AMD3100 versus pla-

cebo-treated cats (P < .05) as well as in cats receiving PMEA

and AMD3100 versus cats receiving PMEA only (P < .05).

Fig 3. Changes in red blood cells (in 106 cells/µL) during the

6-week treatment in cats receiving placebo, AMD3100, PMEA,

or AMD3100 and PMEA. Bars demonstrate the red blood cell

count on day 0, day 14, day 28, and day 42 of all cats in the

respective groups. There was a significant difference between cats

receiving PMEA versus cats receiving placebo (P < .05) and cats

receiving AMD3100 (P < .05) and in cats receiving both com-

pounds (PMEA + AMD3100) compared to the placebo group

(P < .001) and cats receiving only AMD3100 (P < .001).

AMD3100 Treatment against FIV 487

nature of the FIV strain investigated. However, it is dif-ficult to explain why the combination of AMD3100 withPMEA was not effective in reducing viral load. Hypo-thetically, the immunomodulatory properties ofPMEA33,34 might have led to increased production ofFIV-infected lymphocytes and thereby, annihilated thedecrease in proviral load caused by AMD3100.

In the present study, the number of provirus-containing cells declined with AMD3100 treatment.Therefore, the amount of viral RNA would also beexpected to decline. However, no significant change inRNA was detected, even though the absolute viralload in the AMD3100-treated cats of both groupsdecreased. It is possible that treatment with AMD3100for only 6 weeks is insufficient to produce a signifi-cant effect on new viral particle formation, and longertreatment duration would be necessary. However, thisstudy design was chosen because relatively short treat-ment periods with highly effective antiviral combina-tions have been demonstrated to lead to significantimprovement in HIV-infected people.16

Unfortunately the antiproliferative properties ofPMEA described above likely also contributed to theprogressive decline in RBC counts seen in cats treatedwith PMEA alone or in combination with AMD3100.This is a common adverse effect of some acyclic nucle-oside phosphonates, in particular PMEA.17,29,41,42 Theactive intracellular metabolite of PMEA (PMEApp)not only suppresses the activity of retroviral reversetranscriptases but also of some cellular DNA polyme-rases such as DNA polymerase-a.43 Because of thelife-threatening anemia observed in the PMEA-treatedcats, extended treatment with PMEA at the dose usedhere was not possible. Prolongation of the QT intervalon ECG, with potential for development of arrhyth-mias has been reported as an adverse effect afterrepeated intravenous administration of AMD3100 tohumans.44 However, in the present study, all cats wereregularly examined by auscultation of the heart and nosigns of cardiac arrhythmias were found. Likewise, inan experimental FIV trial, in which a higher dosagewas used, no adverse cardiac effects were reported.39

This suggests that cats do not seem to be susceptibleto potential adverse cardiac effects of AMD3100.Other adverse effects of AMD3100 seen in humans aremild transient gastrointestinal signs and an increasedWBC count and serum magnesium concentration.45 Bycontrast, in the present study, cats receiving AMD3100experienced a significant decrease in serum magnesiumconcentration although they did not show any clinicalsigns referable to this. Intestinal absorption of magne-sium utilizes similar mechanisms as calcium. It is there-fore possible that magnesium partially enters the cellwith calcium. Physiologic signal transduction throughCXCR4 causes a calcium influx into the cell. Bindingof AMD3100 to CXCR4 has partial agonistic effects,28

and intracellular calcium concentrations increase pro-portionally with the AMD3100 concentration.28 Thiseffect may have caused magnesium to shift intracellu-larly with a subsequent decline in serum magnesiumconcentrations in the present study. It is unclear,

however, why serum magnesium concentrations areinversely affected in cats and humans. Serum calciumconcentrations did not change significantly in the pres-ent study. To counteract a potential decrease of serumcalcium through influx into cells after binding ofAMD3100 to CXCR4, a fast mobilization of cal-cium from storage sites (ie, bone) is likely to occurimmediately caused by the closely regulated calciumhomeostasis. Initial clinical phase I trials in humanswith AMD3100 showed an unexpected increase in thewhite blood cell counts.14 The drug specificallyincreased the CD34+ hematopoietic stem cell countsin peripheral blood by antagonizing the interaction ofstromal-derived factor 1 (SDF-1) with its CXCR4receptor. However, in cats, no such marked increase ofwhite blood cell counts was observed.

In the present study, no evidence of development ofresistance of FIV against AMD3100 was found. Resis-tance of HIV against AMD3100 could be inducedafter 20–60 passages in the presence of the bicyclamdrug in cell culture.36 The mechanism by which resis-tance developed is believed to be a mutation in the V3region of gp120.46 Perhaps, the treatment period of6 weeks in the present study was not long enough toselect for mutations.

A limitation of the study is the relatively low num-ber of cats per treatment group. However, as for sev-eral variables, a significantly different developmentbetween groups could be demonstrated despite the rel-atively small sample size, the study still provides valu-able information. Based on the results of this study,additional studies including larger numbers of cats andpotentially longer treatment periods are now advisable.

In conclusion, this study evaluated efficacy andadverse effects of the compound AMD3100 againstFIV infection. It was shown that AMD3100 displayeda limited but significant antiviral activity in naturallyFIV-infected cats and did not lead to clinically relevantadverse effects, without observed development of resis-tance during a 6-week treatment period. Because thereare currently very few drugs with proven efficacyagainst FIV, and because most have significant adverseeffects, AMD3100 could be considered as a sole ther-apy or as part of a multidrug treatment. However, useof AMD3100 in combination with PMEA cannot berecommended.

Footnotes

a Mozobil, Genzyme Corporation, Cambridge, MAb Hepsera, Gilead Sciences, Foster City, CAc PetCheck Anti-FIV; IDEXX, Portland, MAd Cell-Dyn 3500; Abbott Laboratories, Abbott Park, ILe Hitachi 911; Roche Deutschland Holding GmbH, Grenzach-

Wyhlen, Germanyf FACS; Becton Dickinson, Heidelberg, Germanyg Cell Quest 1.1.1. and Macintosh Quadra 650 hardware, Apple

Inc, Cupertino, CAh QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany

488 Hartmann et al

i QIAamp Viral Kit; Qiagen GmbHj ABI 7700, Applied Biosystems, Foster City, CAk version 1.6.3., Applied Biosystemsl SuperScript III Platinium One-Step Quantitative RT-PCR

System, Invitrogen, Austriam ABI Prism 7700, Applied Biosystemsn Statistical Package for the Social Sciences, Version 11.5, Inter-

national Business Machines Corporation, Armonk , NY

Acknowledgments

The authors thank K.U. Leuven (GOA 05/19) andUniversity of Utrecht for partial financial support. Wethank Prof Dr David Maggs, Davis, CA, for carefulreading of the manuscript.

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