Inhalation delivery of topotecan is superior to intravenous exposure for suppressing lung cancer in a preclinical model

<p>Intravenous (IV) topotecan is approved for the treatment of various malignancies including lung cancer but its clinical use is greatly undermined by severe hematopoietic toxicity. We hypothesized that inhalation delivery of topotecan would increase local exposure and efficacy against lung cancer while reducing systemic exposure and toxicity. These hypotheses were tested in a preclinical setting using a novel inhalable formulation of topotecan against the standard IV dose. Respirable dry-powder of topotecan was manufactured through spray-drying technology and the pharmacokinetics of 0.14 and 0.79 mg/kg inhalation doses were compared with 0.7 mg/kg IV dose. The efficacy of four weekly treatments with 1 mg/kg inhaled vs. 2 mg/kg IV topotecan were compared to untreated control using an established orthotopic lung cancer model for a fast (H1975) and moderately growing (A549) human lung tumors in the nude rat. Inhalation delivery increased topotecan exposure of lung tissue by approximately 30-fold, lung and plasma half-life by 5- and 4-folds, respectively, and reduced the maximum plasma concentration by 2-fold than the comparable IV dose. Inhaled topotecan improved the survival of rats with the fast-growing lung tumors from 7 to 80% and reduced the tumor burden of the moderately-growing lung tumors over 5- and 10-folds, respectively, than the 2-times higher IV topotecan and untreated control (<i>p</i> < .00001). These results indicate that inhalation delivery increases topotecan exposure of lung tissue and improves its efficacy against lung cancer while also lowering the effective dose and maximum systemic concentration that is responsible for its dose-limiting toxicity.</p

Methylation of <i>TOX2</i> promoter CpG island.

<p><b>A</b>) Combined bisulfite modification and restriction analysis (COBRA) depicts methylation of <i>TOX2</i> promoter CpG island in normal and cancer samples. Complete, partial, or no methylation could be seen from digestion of all, some, or none of the PCR products in the presence of the <i>BstU1</i> (+) enzyme compared to no enzyme (−) control. MDA-MB-231 and MDA-MB-435 in all the figures are abbreviated as M-231 and M-435, respectively. <b>B</b>) Bisulfite sequencing was used to validate methylation results obtained through COBRA and MSP assays and to determine the degree and distribution of methylation at 51 CpG sites across <i>TOX2</i> promoter CpG island. Five clones were sequenced per sample and methylation status of each clone (1/5^th of a circle) at the specified CpG site is shown as methylated (filled) or unmethylated (open).</p

Genome-wide impact of epigenetic inactivation of <i>TOX2</i> and <i>TOX3</i>.

<p>Transfection of (<b>A</b>) <i>Calu-3</i> and <i>MDA-MB-231 (M-231)</i> with siRNAs targeting <i>TOX2</i> (<i>siTOX2</i>) or (<b>B</b>) <i>Calu-3</i> and <i>MCF-7</i> targeting <i>TOX3</i> (<i>siTOX3</i>) reduced expression of these genes by 70–86% compared to cells transfected with control siRNA (siControl). (<b>C and D</b>) However, knockdown of these genes did not change the migration potential of these cells. Genome-wide gene expression assays comparing <i>Calu-3</i> cells transfected with (<b>E</b>) siControl vs. siTOX2 or (<b>F</b>) siControl vs. siTOX3 revealed genes and pathways modulated by epigenetic inactivation of these genes.</p

<i>TOX2</i> expression in normal and cancer cells.

<p>(<b>A</b>) Genomic structure of <i>TOX2</i>. Top box: Predicted transcript variants of <i>TOX2</i> (var.1-4) currently used as reference sequence for <i>Homo sapiens</i> chromosome 20, GRCh37.p2, (GenBank accession number NC_000020.10). Bottom box: Transcripts sequenced from human cells (var.5 and 6). Small arrows indicate the location and direction of primer binding sites; T#F or T#R (forward or reverse primers for TaqMan assays) and G#F or G#R (forward or reverse primers for gel-based assays). (<b>B</b>) Expression of <i>TOX2</i> transcript variants 5 and 6 and the house keeping gene beta-actin in distant normal lung tissue (DNLT), HBEC, and various lung and breast cancer cell lines. In Vehicle-treated (S, for sham) lung cancer (H1838, H2009) and breast cancer (T47D) cell lines with methylated promoter CpG island, both transcripts were silenced and expression of both was primarily restored with 5-Aza-2′-deoxycytidne (D) but not trichostatin A (T) treatment. (<b>C and D</b>) TaqMan assays that use distinct primer sets from those used for gel-based assays confirmed results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034850#pone-0034850-g002" target="_blank">Figure 2B</a>. (<b>C</b>) Expression of TOX2 var.5 or both (var.5 & 6) in lung tumors (n = 20) relative to DNLT (n = 10) obtained from NSCLC patients. (<b>D</b>) Expression of <i>TOX2</i> var.5 or both (var.5+6) in TSA or DAC treated lung and breast cancer cell lines relative to Vehicle-treated (Sham) cell lines.</p

Relative expression of TOX subfamily genes in normal lung tissue.

<p>(<b>A</b>) Expression of each gene was quantified using TaqMan assays and the level of <i>TOX4</i>, which is unmethylated in all samples and expressed the highest in normal lung tissue, was used as a reference to calculate the relative level of the remaining genes. * p = 0.03, ** p<0.001, *** p<0.0001 compared to <i>TOX4</i>. (<b>B</b>) COBRA conducted as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034850#pone-0034850-g001" target="_blank">Figure 1A</a>. (<b>C</b>) <i>TOX</i> expression was measured relative to its expression in <i>MCF-7</i> (Top left) or vehicle treated <i>MDA-MB-231 (M-231)</i>, <i>T47D</i>, or <i>MCF-7</i>. (<b>D</b>) Transfection of <i>M-231</i> with siTOX reduced its expression by 75% compared to siControl (left) but this did not alter the migration potential of the cells.</p

Prevalence for promoter CpG island hypermethylation of <i>TOX</i> subfamily of genes.

A<p>Methylation of <i>TOX</i> was significantly more prevalent in breast than lung tumor (p<0.001). In contrast, <i>TOX3</i> methylation was more common in lung than breast tumor (p<0.001).</p>B<p>Among NSCLC patients, the prevalence for <i>TOX2</i> methylation in current smokers was significantly higher than never smokers (p<0.05) as well as current non-smokers (former and never smokers combined) (p<0.05).</p>C<p><i>TOX3</i> methylation in primary lung tumors was marginally more prevalent in never smokers compared to current or former smokers (p = 0.05).</p>D<p><i>TOX3</i> methylation in primary lung tumors was more prevalent in squamous cell carcinoma compared to adenocarcinoma (p = 0.05).</p