Colchicine Reduces Inflammation in a Humanized Mouse Model of Sickle Cell Disease

Introduction Inflammation, marked by a "cytokine storm" of circulating proinflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor- α (TNF-α), and C-reactive protein (CRP), and increases in intracellular NLRP3 inflammasomes is an important component of sickle cell disease (SCD). Although current treatments have limited anti-inflammatory effects, targeting inflammation is a promising but unrealized treatment approach.

Colchicine, a tubulin inhibitor, is a promising anti-inflammatory agent for SCD. It is useful in gout, familial Mediterranean fever, pericarditis and other inflammatory diseases. In these contexts, it often reduces the same inflammatory proteins and cellular components that are elevated in SCD.

We sought preliminary evidence for colchicine as an anti-inflammatory treatment for SCD.

Methods Humanized sickle mice (HbSS-BERK) are knockout for murine α and β globins and express human α and β^S globins (>99% β^S) on a mixed genetic background. HbSS-BERK have severe disease including hyperalgesia. Experiments were performed following protocols approved by the Institutional Animal Care and Use Committee.

Female mice of ~ 3.5 months of age received either colchicine 100 µg/kg body weight or vehicle (phosphate-buffered saline) intraperitoneally daily for 14 d. Pain-related behavior testing was performed before treatment and 1 h, 24 h, 7 d, and 14 d after the 1^st dose of study agent. Mechanical hyperalgesia was tested by paw withdrawal frequency (PWF) in response to the application of a von Frey monofilament to the mid-plantar surface of the hind paw. Musculoskeletal/deep tissue hyperalgesia was tested by measurement of the peak tensile force exerted by a forelimb upon traction. Cold hyperalgesia was tested by PWF/2 min upon placement of mice on a cold plate maintained at ~4°C.

After the 14 d of dosing, mice were euthanized; blood was collected; mice were necropsied; and 4 mm diameter punch biopsies of dorsal skin were harvested and incubated in culture medium for 24 hours to allow for release of proteins into the skin secretagogue Plasma serum amyloid-P (SAP) and IL-6 were analyzed with enzyme-linked immunosorbent assays. Limited blood volumes prevented assay of other circulating proteins. Automated and manual cytometry were used to characterize circulating blood cells. Skin secretagogue was assayed for cytokines IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, monocyte chemoattractant protein 1 (MCP-1), interferon gamma (IFN-γ), TNF-α, macrophage inflammatory protein-1 alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulated on activation, normal T-cell expressed and secreted protein (RANTES) using Q-Plex^TM microarray. Data was analyzed using conventional descriptive statistics.

Results As per usual laboratory practice, we considered mouse circulating SAP levels as surrogate for human CRP levels. Following 14 d of dosing, plasma IL-6 levels were ~67% lower (~130 v ~48 µg/g protein; p<0.01), and plasma SAP levels were ~45% lower (~33 v ~18 µg/g protein; p<0.01) in colchicine-treated mice. There were no significant differences in blood cell parameters. Concentrations of IL-3, IL-1, IFN-γ, and GM-CSF were 40% (p<0.05), 20% (p<0.05), 40% (p<0.01), and 40% (p<0.01) lower, respectively, in the skin secretagogue of colchicine-treated mice. Given the chronic nature of inflammation in SCD, we did not anticipate phenotypic changes after only a 14-day exposure to colchicine. Indeed, no significant differences in hyperalgesia or mouse/organ weight were observed.

We think that next steps should be dose-finding studies of colchicine in adults living with SCD followed by randomized, placebo-controlled trials with clinically relevant endpoints such as repeated measures of pain interference using a validated instrument. The anticipated lack of colchicine effect upon blood cell parameters suggests that patients would benefit more from a combination of a disease-modifying agent such as hydroxyurea in combination with colchicine.

Argueta:Cyclerion: Honoraria; Cayenne Foundation: Honoraria. Gupta:UCI Foundation: Research Funding; Zilker LLC: Research Funding; Grifols: Research Funding; 1910 Genetics: Research Funding; Cyclerion: Research Funding; CSL Behring: Honoraria; Novartis: Honoraria, Research Funding; Tautona Group: Honoraria; SCIRE Foundation: Research Funding. Roberts:Pfizer: Consultancy; Truven Health Analytics: Consultancy; Community Health Network of Connecticut: Consultancy; Magellan Rx: Consultancy.

We will propose investigating colchicine as an anti-inflammatory agent for the treatment of the inflammation of sickle cell disease.