Adverse Events.

<p>Adverse Events.</p

Phase 1 Study of a Sulforaphane-Containing Broccoli Sprout Homogenate for Sickle Cell Disease

<div><p>Sickle cell disease (SCD) is the most common inherited hemoglobinopathy worldwide. Our previous results indicate that the reduced oxidative stress capacity of sickle erythrocytes may be caused by decreased expression of NRF2 (Nuclear factor (erythroid-derived 2)-like 2), an oxidative stress regulator. We found that activation of NRF2 with sulforaphane (SFN) in erythroid progenitors significantly increased the expression of NRF2 targets <i>HMOX1</i>, <i>NQO1</i>, and <i>HBG1</i> (subunit of fetal hemoglobin) in a dose-dependent manner. Therefore, we hypothesized that NRF2 activation with SFN may offer therapeutic benefits for SCD patients by restoring oxidative capacity and increasing fetal hemoglobin concentration. To test this hypothesis, we performed a Phase 1, open-label, dose-escalation study of SFN, contained in a broccoli sprout homogenate (BSH) that naturally contains SFN, in adults with SCD. The primary and secondary study endpoints were safety and physiological response to NRF2 activation, respectively. We found that BSH was well tolerated, and the few adverse events that occurred during the trial were not likely related to BSH consumption. We observed an increase in the mean relative whole blood mRNA levels for the NRF2 target <i>HMOX1</i> (p = 0.02) on the last day of BSH treatment, compared to pre-treatment. We also observed a trend toward increased mean relative mRNA levels of the NRF2 target <i>HBG1</i> (p = 0.10) from baseline to end of treatment, but without significant changes in HbF protein. We conclude that BSH, in the provided doses, is safe in stable SCD patients and may induce changes in gene expression levels. We therefore propose investigation of more potent NRF2 inducers, which may elicit more robust physiological changes and offer clinical benefits to SCD patients.</p><p><b><i>Trial Registration</i>:</b> ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT01715480" target="_blank">NCT01715480</a></p></div

Fetal hemoglobin levels during study period.

<p>Fetal hemoglobin levels were assessed with hemoglobin electrophoresis for the A) 50g, B) 100g, and C) 150g cohorts. Each individual patient is designated with letter, symbol, and color codes; several patients participated in multiple cohorts. Patient F was diagnosed with HbSß° thalassemia; all other patients were diagnosed with HbSS. Patients taking hydroxyurea during the study period are denoted with an asterisk.</p

CONSORT flow diagram enrollment summary

<p>CONSORT flow diagram enrollment summary</p

Clinical Parameters.

<p>Clinical Parameters.</p

Effect of BSH ingestion on whole blood mRNA in SCD subjects.

<p>The composite of the mean for relative mRNA expression of A) NQO1, B) HMOX1, and C) HBG1/(HBB+HBG1) for all patients during the study period. All expression was set relative to pre-treatment values. Expression was normalized with GAPDH, and all statistical analyses were performed using Friedman’s test (* = p<0.05).</p

Average percent change of mRNAs at end treatment relative to pre-treatment.

<p>Average percent change of mRNAs at end treatment relative to pre-treatment.</p

<i>apol1</i> interacts with <i>myh9</i> in an anemic context.

<p>To test for epistatic effects of <i>apol1</i> and <i>myh9</i> in zebrafish, we first co-injected both <i>apol1-</i>MO (1.0ng/nl dose) and <i>myh9-</i>MO (6.0ng/nl dose) into zebrafish larvae and scored for edema formation at 5 dpf. (n = 39–89 embryos/injection; repeated three times). However, under this co-suppression model (A, B), we observed no significantly increased edema formation compared to each MO alone. We next tested for an interaction between <i>apol1</i> and <i>myh9</i> in the context of <i>atpif1a</i> suppression, predicting that the added stress of anemia would mimic our initial observations in sickle cell disease patients. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hours later. (C) Representative eye image series of zebrafish embryos for each injection group show relatively stable or decreased fluorescence intensity over time. (E) Bar graphs summarize the changes observed for each injection group. Zebrafish embryos injected with all three MOs show a significant increase in dextran clearance from the vasculature compared to co-suppression of <i>apol1</i> and <i>myh9</i>. (D, F) These data are reproduced using butafenacil induced anemia (0.195 μM in embryo media, treated at 48 hpf). Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (<i>n =</i> 19); <i>atpif1a</i> MO injected (<i>n =</i> 14); <i>apol1-</i>MO+<i>myh9</i>-MO (<i>n =</i> 12); <i>apol1-</i>MO+<i>myh9</i>-MO+<i>atpif1a</i>-MO (n = 11); Butafenacil (n = 48); But+<i>myh9-</i>MO+<i>apol1-</i>MO (n = 18). hpf, hours post-fertilization; hpi, hours post-injection. *p<0.001.</p

Comparison of APOL1 human and zebrafish protein sequences and relevance to the zebrafish kidney.

<p>Protein domain schematic of (A) zebrafish APOL1 and (B) human APOL1 is shown, with zebrafish domains (NP_001025309) aligned to the human protein (NP_001130012) and coded based on summarized consensus scores (Gonnet PAM 250 matrix, Clustal Omega, Cambridge, UK; <i>S</i>, secretory domain, <i>PFD</i>, pore-forming domain, <i>B</i>, BH3 domain, <i>MAD</i>, membrane-addressing domain, <i>SRA</i>, serum resistance-associated binding domain). Prominent regions of the human and zebrafish alignments are expanded, including the (C) BH3 domain and (D) SRA binding domain, and consensus symbols are displayed (* (asterisk), fully conserved;: (colon), >0.5 in the Gonnet PAM 250 matrix;. (period), = <0.5 in the Gonnet PAM 250 matrix). The leucine zipper domain (codons 365–392 in <i>APOL1</i>, underline), and the location of the G1 and G2 risk alleles in CKD in African Americans (S342G/I384M and ΔN388Y389) are highlighted in red. (E) Podocytes from adult glomeruli of <i>pod</i>::NTR-mCherry zebrafish were flow-sorted and evaluated for <i>apol1</i> RNA expression through RT-PCR. <i>apol1</i> is expressed in fluorescence-activated cell sorted (FACS) podocytes and the adult liver. FACS podocytes also express zebrafish <i>podocin</i> (<i>nphs2)</i> but a purkinje-cell marker, <i>wdr81</i>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005349#pgen.1005349.ref029" target="_blank">29</a>], was undetectable. NT = non-template reverse transcription control; L = dissected adult liver cells from <i>pod</i>::NTR-mCherry zebrafish; P = fluorescence-activated cell sorted podocytes from dissected glomeruli of <i>pod</i>::NTR-mCherry zebrafish; Em = 5 dpf whole-zebrafish embryo cDNA.</p

<i>apol1</i> morphant zebrafish embryos display generalized edema and glomerular filtration defects indicative of nephropathy.

<p>Representative live images of (A) sham-injected control larvae, and (B) <i>apol1</i> morpholino (MO) injected larvae at 5 dpf. <i>apol1</i> morphants display pericardial and yolk sac edema. (C) Injection of increasing doses of <i>apol1</i>-MO demonstrate dose-dependent effects when scored for generalized edema (<i>n</i> = 35–65 embryos/injection; repeated three times) compared to control larvae at 5 dpf. <i>apol1</i> morpholino injected embryos were complemented with the respective human mRNA to <i>APOL1</i> (100pg/nl) and scored for generalized edema at 5 dpf. (D) Ectopic expression of <i>APOL1</i> rescues significantly the edema phenotype observed in <i>apol1</i> morphants (1.0 ng/nl dose). We observed no significant phenotypes when <i>APOL1</i> human mRNA is injected alone. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hpi. (E) Representative eye image series of zebrafish larvae for each injection group show a relatively stable or a decrease in fluorescence intensity over time compared to sham-injected controls. (F) Bar graphs summarize the fluorescence changes observed for each injection group for <i>apol1</i> morphant larvae. Reduction in fluorescence intensity over the pupil was calculated relative to the 24 hpi time point; <i>apol1</i> morphants display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, indicative of compromised glomerular filtration and proteinuria. These defects were rescued significantly when MO was co-injected with orthologous human mRNA. (G-I) Compared to (G) sham-injected controls, the glomerular ultrastructure of (H) <i>apol1</i> morphant zebrafish display partial effacement of podocyte foot process (* asterisks), although the glomerular basement membrane (filled arrowheads) appears normal. Microvillus protrusions (open arrowheads) are also apparent in the urinary space. (I) Ultrastructure defects are rescued upon co-injection of human wild-type mRNA (100pg). Scale bar, 500nm. White bars, normal; black bars, edema. MO concentrations are in μg/μl, with 1nl injected into each embryo. C, sham-injected control; NI, non-injected control. Dextran values are in relative fluorescent intensity, mean ± SE. Control, sham-injected control (<i>n =</i> 29); MO, <i>apol1</i> morpholino injected (<i>n =</i> 26); <i>apol1-</i>MO+mRNA (<i>n =</i> 28). h.p.f., hours post-fertilization; h.p.i., hours post-injection. *p<0.001.</p