Evaluation of the safety and immunomodulatory effects of sargramostim in a randomized, double-blind phase 1 clinical Parkinson's disease trial

Abstract

A potential therapeutic role for immune transformation in Parkinson's disease evolves from more than a decade of animal investigations demonstrating regulatory T cell (Treg) nigrostriatal neuroprotection. To bridge these results to human disease, we conducted a randomized, placebo-controlled double-blind phase 1 trial with a well-studied immune modulator, sargramostim (granulocyte-macrophage colony-stimulating factor). We enrolled 17 age-matched non-Parkinsonian subjects as non-treated controls and 20 Parkinson's disease patients. Both Parkinson's disease patients and controls were monitored for 2 months for baseline profiling. Parkinson's disease patients were then randomized into two equal groups to self-administer placebo (saline) or sargramostim subcutaneously at 6 μg/kg/day for 56 days. Adverse events for the sargramostim and placebo groups were 100% (10/10) and 80% (8/10), respectively. These included injection site reactions, increased total white cell counts, and upper extremity bone pain. One urticarial and one vasculitis reaction were found to be drug and benzyl alcohol related, respectively. An additional patient with a history of cerebrovascular disease suffered a stroke on study. Unified Parkinson's disease rating scale, Part III scores in the sargramostim group showed modest improvement after 6 and 8 weeks of treatment when compared with placebo. This paralleled improved magnetoencephalography-recorded cortical motor activities and Treg numbers and function compared with pretreated Parkinson's disease patients and non-Parkinsonian controls. Peripheral Treg transformation was linked to serum tryptophan metabolites, including L-kynurenine, quinolinic acid, and serotonin. These data offer a potential paradigm shift in modulating immune responses for potential therapeutic gain for Parkinson's disease. Confirmation of these early study results requires larger numbers of enrolled patients and further clinical investigation.

Conflict of interest statement

All authors indicate no competing interests.

Figures

Fig. 1

Peripheral blood lymphocytes from PD patients treated with placebo or sargramostim were assessed for the expression of Treg phenotype and function over a 3-month mean baseline (visits 1–3), every 2 weeks after the initiation of treatment (visits 4–7), and 4 weeks after discontinuation of treatment (visit 8). Flow cytometric analyses for percentage of a CD4+ Teffs (CD4+CD127hiCD25hi), b CD4+ Tregs (CD4+CD127loCD25hi), c FOXP3+CD4+ Tregs, d iCTLA4+CD4+ Tregs, e CD39+CD4+ Tregs, and f FAS+CD4+ Tregs. Plots represent the medians, interquartile ranges (IQRs) (boxes), and non-outlier ranges (whiskers) for T cells from PD patients. Levels of T cell subsets from PD patients treated with placebo (n = 6–10) (blue) or sargramostim (n = 5–9) (red) were compared by Mann–Whitney U test with P ≤ a0.10, b0.05, or c0.01. g–i Enriched Treg isolates were assessed for the capacity to suppress CD3/CD28-stimulated CD4+CD25− Tresps from a healthy donor. Tregs were serially diluted two-fold and co-stimulated with a constant number of CFSE-stained Tresps to yield decreasing Treg:Tresp ratios. Treg activity as percentage inhibition of proliferation was determined for g non-Parkinsonian controls (n = 17) and non-allocated PD patients (n = 20) at 8, 4, and 0 weeks before treatment initiation (Entry, visits 1–3); h for randomized PD patients prior to initiation of treatment (Pre-Treatment, visits 1–3); and i at 2, 4, 6, and 8 weeks after initiation (Treatment, visits 4–7). Comparison of differences in slope or elevation as an indicator of Treg activity was determined by linear regression analyses for baseline paired controls and PD patients (Pslope = 0.49, Pelevation = 0.065, n = 17) (Entry); for baseline of placebo (n = 10) or sargramostim (n = 10) randomized PD patients (Pslope = 0.59, Pelevation = 0.17) (Pre-Treatment); and for PD patients during treatment with sargramostim (n = 5–9) compared with placebo (n = 9–10) (Pslope = 0.063, Pelevation = 0.058) (Treatment). Comparison of Treg activity from pre-treated and treated patients randomized to sargramostim group (Pslope = 0.039) or placebo group (Pslope = 0.88, Pelevation = 0.04)

Fig. 2

Metabolomic analyses from serum from sargramostim-treated or placebo-treated PD patients. a Global metabolomic analysis was performed on serum samples from the entire PD patient cohort. Comparisons of metabolic features were performed between sample groups specifically focusing on pre-treatment groups (visits 1 and 2) vs. on treatment groups (visits 5 and 7), and between treatment groups either on placebo or on sargramostim. A cloud plot illustrating the dysregulated features between pre-treatment and on-treatment are overlaid on the chromatographic runs. Each circle represents a dysregulated feature at a specific retention time (x-axis) and mass-to-charge ratio (y-axis). The diameter of each feature represents the fold change and the color intensity represents the significance (P-value). Six-hundred metabolites were found to be upregulated or downregulated in PD patients treated with sargramostim compared with their respective pre-treated controls. b The 600 dysregulated metabolites were cross-referenced with known metabolic pathways, analyzed by Welch’s t-test to identify dysregulated features, and altered metabolic pathways were determined by using the mummichog algorithm which maps possible metabolite matches and targets local enrichments that reflect true pathway activity opposed to false matches that otherwise are randomly distributed. The plot shows the statistical relevance of dysregulated metabolic pathways for sargramostim-treated patients compared with pre-treated controls as the −log10P-value as the function of the weighted mean percentage overlap of metabolite pathway identifying the tryptophan metabolism as a key pathway affected by treatment with sargramostim. The greater color intensity represents a more significant P-value and the diameter represents the percent coverage of metabolites found to be dysregulated in a given pathway. c Targeted metabolomic analyses of serum from PD patients at pretreatment (Pre, visits 1 and 2), at weeks 4 and 8 during treatment (On, visits 5 and 7), and at 4 weeks after treatment cessation (Post, visit 8). When available, results from the same patient, but at different visits were averaged and binned into pre-treatment or on-treatment. Medians and IQRs of tryptophan metabolite concentrations were determined from patients randomized into placebo group (blue bars) (nPre = 8, nOn = 9, nPost = 8) or sargramostim group (red bars) (nPre = 9, nOn = 7, nPost = 5). Comparison of median metabolite concentrations between pre-treatment, on-treatment, and post-treatment samples and between samples from placebo-treated and sargramostim-treated groups were determined by Mann–Whitney U tests. Of the 18 targeted metabolites from the tryptophan pathway, many were below the calibration curve or detection limits, or were unchanged. d Metabolomic analysis showed concentrations of kynurenine and quinolinic acid upregulated, whereas serotonin was downregulated within the tryptophan pathway. Enzymes in the tryptophan pathway include TPH2, tryptophan hydroxylase-2; 5HTD, 5-hydroxytryptophan decarboxylase; TDO, tryptophan 2,3-dioxygenase; IDO, indoleamine 2,3-dioxygenase; AFMID, arylformamidase; KMO, kynurenine 3-monooxygenase; KYNU, kynureninase; and HAO, 3-hydroxyanthranilate 3,4-dioxygenase

Fig. 3

T cell gene expression analyses of T cells from sargramostim-treated or placebo-treated PD patients. a Significant increase or decrease in expression of genes by CD4+CD25− T cells from PD patients treated with sargramostim compared with placebo. Genes are divided into those associated with Th1 and Th17 (Pro-inflammatory), Th2 and Tregs (Anti-inflammatory), and general T cell proliferation and differentiation (Non-associated). Significant differences are indicated by a heat map. The map ranged from 40-fold increase (red) to 40-fold decrease (green). b Ingenuity pathway analyses performed on upregulated or downregulated genes to identify putative network associations involved in hematological development and T cell function. Genes and mediators that are upregulated are shaded red with the darker shades indicating more upregulation; shades of green denote downregulation; and nodes in white represent putative-associated function

Fig. 4

PD patients were randomized to receive placebo or sargramostim. a UPDRS III scores of each individual patient were assessed at 0, 4, and 8 weeks (visits 1–3) before treatment (Pre-Treatment); at 2, 4, 6, and 8 weeks (visits 4–7) during treatment (Placebo or Sargramostim); and at 4 weeks (visit 8) after cessation (Placebo or Sargramostim Post-Treatment). Higher scores represent more severe motor symptoms. b Changes from baseline UPDRS III scores were determined at each visit for placebo-treated and sargramostim-treated patients using the mean scores of visits 1–3 for each patient as baseline from which to normalize. Changes in scores from each randomized treatment group were normally distributed and homoscedastic by Levene’s test (P > 0.05). Factorial ANOVA showed an effect of randomized treatment group (P = 0.05) and marginal effects of visit (P = 0.07) and treatment-by-time (P = 0.05). Fisher’s least significant difference post hoc tests were used to determine pairwise differences between placebo and sargramostim treatment at each visit. UPDRS III scores for placebo group diminished at start of treatment, which may reflect a placebo effect, but returned to baseline during the study course. c MEG assessment of beta ERD in PD patients. Paired sample t-test comparison of beta ERD activity at baseline (pre-treatment) and during treatment for the group of PD patients receiving sargramostim. Significant increases in beta ERD amplitudes are noted for the pre-treated patient composite in the left and right precentral gyri, right premotor cortex, and SMA (top panel). Increases in beta ERD activity from pre-treatment to sargramostim-treatment are shown for individual patients. Compared with pre-treatment, the left precentral gyrus showed a significant effect of visit [F(2, 9) = 8.869, P = 0.007] and visit-by-group interaction [F(2, 9) = 6.04, P = 0.022], which was quadratic [F(1, 10) = 10.772, P = 0.008]. The right precentral gyrus also showed a visit-by-group interaction [F(2, 9) = 3.321, P = 0.06], which also was quadratic [F(1, 10) = 5.447, P = 0.04]. The right premotor cortex showed a marginal effect of visit [F(2, 9) = 3.050, P = 0.07] and the effect was quadratic [F(1, 10) = 6.124, P = 0.03]. Quadratic interactions were explained as beta ERD amplitudes that increase from pretreatment baseline while on sargramostim and return to baseline levels after termination of treatment