LD-Aminopterin in the Canine Homologue of Human Atopic Dermatitis: A Randomized, Controlled Trial Reveals Dosing Factors Affecting Optimal Therapy

<div><p>Background</p><p>Options are limited for patients with atopic dermatitis (AD) who do not respond to topical treatments. Antifolate therapy with systemic methotrexate improves the disease, but is associated with adverse effects. The investigational antifolate LD-aminopterin may offer improved safety. It is not known how antifolate dose and dosing frequency affect efficacy in AD, but a primary mechanism is thought to involve the antifolate-mediated accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). However, recent <i>in vitro</i> studies indicate that AICAR increases then decreases as a function of antifolate concentration. To address this issue and understand how dosing affects antifolate efficacy in AD, we examined the efficacy and safety of different oral doses and schedules of LD-aminopterin in the canine model of AD.</p><p>Methods and Findings</p><p>This was a multi-center, double-blind trial involving 75 subjects with canine AD randomized to receive up to 12 weeks of placebo, once-weekly (0.007, 0.014, 0.021 mg/kg) or twice-weekly (0.007 mg/kg) LD-aminopterin. The primary efficacy outcome was the Global Score (GS), a composite of validated measures of disease severity and itch. GS improved in all once-weekly cohorts, with 0.014 mg/kg being optimal and significant (43%, <i>P</i><0.01). The majority of improvement was seen by 8 weeks. In contrast, GS in the twice-weekly cohort was similar to placebo and worse than all once-weekly cohorts. Adverse events were similar across all treated cohorts and placebo.</p><p>Conclusions</p><p>Once-weekly LD-aminopterin was safe and efficacious in canine AD. Twice-weekly dosing negated efficacy despite having the same daily and weekly dose as effective once-weekly regimens. Optimal dosing in this homologue of human AD correlated with the concentration-selective accumulation of AICAR <i>in vitro</i>, consistent with AICAR mediating LD-aminopterin efficacy in AD.</p></div

Subject demographics and baseline AD characteristics.

<p>Abbreviations: GS, Global Score; CADESI, Canine Atopic Dermatitis Extent and Severity Index 03; PVAS, Pruritus Visual Analogue Scale.</p><p>Data are mean ± SD for continuous variables.</p>a<p><i>P</i>-values were calculated by chi-square test for categorical data and one-way ANOVA for continuous data.</p><p>Subject demographics and baseline AD characteristics.</p

Effect of placebo and LD-aminopterin on canine AD disease measures.

<p>Subjects (<i>N</i> = 75) with AD were randomized equally to receive placebo, or LD-aminopterin once-weekly (0.007, 0.014 or 0.021 mg/kg) or twice-weekly (0.007×2 mg/kg). Improvement in baseline disease measures were determined for (A) GS, (B) PVAS and (C) CADESI (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#s2" target="_blank">Materials and Methods</a>). GS and PVAS improved significantly in the 0.014 mg/kg cohort. *<i>P</i><0.05. Horizontal bars are medians. Abbreviations: GS, Global Score; PVAS, Pruritus Visual Analogue Scale; CADESI, Canine Atopic Dermatitis Extent and Severity Index 03.</p

Summary of clinical AEs by cohort^a.

<p>Abbreviations: AE, adverse event.</p>a<p>Expressed as <i>n</i> and percent of total subjects in each cohort.</p>b<p>AE led to discontinuation by subject owner, not by investigator.</p>c<p>0.007×1 mg/kg cohort: <i>Demodex canis</i> at day 44 post 0.5 mg/kg prednisolone on days 0 to 14; and 0.007×2 mg/kg cohort: <i>Demodex injai</i> at day 56 post 1.0 mg/kg prednisolone on days 0 to 14. Demodicosis cleared after one dose of milbemycin oxime, and each subject treated with LD-aminopterin for 24 (0.007×1 mg/kg cohort) and 9 (0.007×2 mg/kg cohort) months in the open-label segment without recurrence.</p><p>Summary of clinical AEs by cohort<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#nt107" target="_blank">^a</a>.</p

LD-Aminopterin composition and mechanistic model in anti-inflammation.

<p>(A) Chemical structure of L-aminopterin (<i>top</i>) and D-aminopterin (<i>bottom</i>). (B) The anti-inflammatory activity of L-aminopterin and methotrexate have been attributed to inhibition of thymidylate (<i>red</i>) and purine (<i>green</i>) <i>de novo</i> biosynthesis. In the <i>de novo</i> pathway of thymidylate (dTMP) synthesis, serine hydroxymethyltransferase (SHMT) catalyzes the conversion of serine and tetrahydrofolate polyglutamates (THF) to 5,10-CH₂-THF and glycine. Thymidylate synthase (TYMS) converts 5,10-CH₂-THF and deoxyuridine monophosphate (dUMP) to dihydrofolate polyglutamates (DHF) and dTMP. Dihydrofolate reductase (DHFR) completes the cycle by catalyzing the conversion of DHF to THF in an NADPH-dependent reaction. The purine, inosine monophosphate (IMP), is synthesized <i>de novo</i> in 10 chemical steps (shown numbered) catalyzed by six enzymes. The six enzymes are phosphoribosylpyrophosphate amidotransferase (PPAT; 1); a trifunctional enzyme composed of glycinamide ribonucleotide synthetase (GARS; 2), GAR formyltransferase (GART; 3) and aminoimidazole ribonucleotide synthetase (AIRS; 5); formylglycinamidine ribonucleotide synthase (FGAMS; 4); a bifunctional enzyme composed of carboxyaminoimidazole ribonucleotide synthase (CAIRS; 6) and succinoaminoimidazolecarboxamide ribonucleotide synthetase (SAICARS; 7); adenylosuccinate lyase (ASL; 8); and a bifunctional enzyme composed of aminoimidazolecarboxamide ribonucleotide transformylase (AICART; 9) and inosine monophosphate cyclohydrolase (IMPCH; 10). Evidence indicates that 10-formyl-7,8-dihydrofolate (10-CHO-DHF) is the predominant <i>in vivo</i> substrate for AICART, making AICART and TYMS the only enzymes to produce the DHFR substrate DHF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#pone.0108303-Baggott4" target="_blank">[69]</a>. Inside the cell, L-aminopterin and methotrexate and their polyglutamate metabolites (antifol) bind with high affinity to DHFR, resulting in accumulation of DHF and depletion of the reduced folate pool. Depletion of folates, as well as the direct inhibition by antifol and DHF, have all been implicated in the inhibition of PPAT, GART, AICART and TYMS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#pone.0108303-Sant1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#pone.0108303-Allegra2" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#pone.0108303-Lyons1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#pone.0108303-Seither1" target="_blank">[70]</a>. In the case of AICART, the accumulation of DHF may cause this reaction to run backwards, since AICAR is normally driven towards the biosynthesis of FAICAR and IMP by the DHFR-catalyzed reduction of DHF to THF, as the equilibrium of this step actually lies in the direction of AICAR formation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#pone.0108303-Wall1" target="_blank">[60]</a>.</p

Summary of laboratory AEs by cohort^a.

<p>Abbreviations: RBC, red blood cell; BUN, blood urea nitrogen; ALT, alanine transaminase.</p>a<p>Expressed as <i>N</i> and percent of 75 total subjects.</p><p>Summary of laboratory AEs by cohort<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108303#nt111" target="_blank">^a</a>.</p

Concomitant medications.

a<p><i>P</i>-values calculated by chi-square test.</p>b<p>During first 14 days.</p><p>Concomitant medications.</p

Study flow chart.

<p>Randomized subjects with AD were orally administered placebo, or LD-aminopterin once-weekly (0.007×1 mg/kg, 0.014×1 mg/kg, 0.021×1 mg/kg) or twice-weekly (0.007×2 mg/kg).</p

Disposition of subjects.

<p>A total of 68 subjects (91%) completed the study per protocol. Discontinuations (9%) were for withdrawal of owner consent (<i>N</i> = 2), owner perceived AE (<i>N</i> = 2), and prohibited medication (<i>N</i> = 1).</p