Researchers at the University of California, Berkeley, have found that a drug once widely used to wean alcoholics off of drinking helps to improve sight in mice with retinal degeneration.
A group of scientists led by Richard Kramer, UC Berkeley professor of molecular and cell biology, had previously shown that a chemical -retinoic acid – is produced when light-sensing cells in the retina, called rods and cones, gradually die off.
This chemical causes hyperactivity in retinal ganglion cells, which ordinarily send visual information to the brain. The hyperactivity interferes with their encoding and transfer of information, obscuring vision.
He realized, however, that the drug disulfiram – also called Antabuse – inhibits not only enzymes involved in the body’s ability to degrade alcohol, but also enzymes that make retinoic acid. In new experiments, Kramer and collaborator Michael Goard, who directs a lab at UC Santa Barbara (UCSB), discovered that treatment with disulfiram decreased the production of retinoic acid and made nearly-blind mice much better at detecting images displayed on a computer screen.
Kramer suspects that retinoic acid plays an identical role in people with vision loss. But experiments measuring retinoic acid in the eye have not been done on humans because they would be too invasive.
Disulfiram – which is already approved for use by the Food and Drug Administration (FDA) – could establish that link. The researchers are planning to partner with ophthalmologists to conduct a clinical trial of disulfiram on patients with RP. The trial would be carried out on a small set of people with advanced, but not yet complete, retinal degeneration.
“There may be a long window of opportunity in which suppressing retinoic acid with drugs like disulfiram could substantially improve low vision and make a real difference in people’s quality of life,” said Kramer, the CH and Annie Li Chair in Molecular Biology of Diseases at UC Berkeley and a member of the campus’s Helen Wills Neuroscience Institute. “Because the drug is already FDA-approved, the regulatory hurdles are low. It wouldn’t be a permanent cure, but right now there are no available treatments that even temporarily improve vision.”
Kramer, Goard and their colleagues – Michael Telias, a former UC Berkeley postdoctoral fellow now at the University of Rochester Medical Center, and Kevin Sit of UCSB – will publish their findings March 18 in the journal Science Advances.
Kramer acknowledged that disulfiram may not be for everyone. When combined with alcohol consumption, the drug can have severe side effects, including headache, nausea, muscle cramps and flushing.
But if disulfiram can improve vision, more targeted therapies could be sought that don’t interfere with alcohol breakdown or other metabolic functions. The researchers have already tested an experimental drug named BMS 493 that inhibits the receptor for retinoic acid, and they have also used an RNA interference technique – a type of gene therapy – to knock down the receptor. Both of these procedures also dramatically improved vision in mice with RP.
Three years ago, Kramer and his colleagues reported that retinoic acid generated sensory noise that interfered with remaining vision in mice with RP in the same way that ringing in the ears, known as tinnitus, can interfere with hearing in people who are losing vibration-sensitive cells in the inner ear. They showed that inhibiting the retinoic acid receptor reduced the noise and increased simple light avoidance behaviors in those mice.
But do mice treated with the drugs actually see better?
The new study provides evidence that they do. First, when the mice were young and had healthy retinas, they were trained to recognize and respond to a simple image of black and white stripes displayed on a computer screen. A month later, after most of the rods and cones had degenerated, the image was shown once again. The investigators found that mice treated with disulfiram or BMS 493 responded quite well, even if the image was blurry. By contrast, mice receiving a placebo failed to respond, even if the image was crisp and clear.
In a second type of study, the scientists used a special microscope and a fluorescent protein indicator to light up and examine the responses of thousands of cells in the brain to much more complex visual scenes—a Hollywood movie clip, replayed many times.
Individual cells in the brains of vision-impaired mice with RP responded preferentially to particular frames in the movie, and their responses were much stronger and more reliable than those of mice that had been treated with disulfiram or BMS 493.
The responses were so reliable, Kramer said, that the investigators could deduce which specific scene had triggered the cell’s response, but only in the mice that had been treated with one of the drugs.
Both the behavioral results and the brain imaging results suggest that the drugs improve vision and not just light detection.
“Treated mice really see better than mice without the drugs. These particular mice could barely detect images at all at this late stage of degeneration. I think that that’s quite dramatic,” Kramer said.
In 2019, Kramer and his team laid out the mechanism behind hyperactivity caused by degeneration. They found that retinoic acid, which is well-known as a signal for growth and development in embryos, floods the retina when photoreceptors – the rods, sensitive to dim light, and the cones, needed for color vision – die.
That’s because photoreceptors are packed with light-sensitive proteins called rhodopsin, which contain retinaldehyde. When the retinaldehyde can no longer be absorbed by rods and cones, it is converted to retinoic acid by an enzyme called retinaldehyde dehydrogenase.
The retinoic acid, in turn, stimulates the retinal ganglion cells by adhering to retinoic acid receptors. It’s these receptors that make ganglion cells hyperactive, creating a constant buzz of activity that submerges the visual scene and prevents the brain from picking out the signal from noise. Drug developers could seek to prevent this by developing chemicals to stop production of retinoic acid by retinaldehyde dehydrogenase, or chemicals that interfere with the retinoic acid receptor.
“If a vision impaired human were given disulfiram, and their vision got better, even a little bit, that would be a great outcome in itself. But it would also strongly implicate the retinoic acid pathway in vision loss,” Kramer said. “And that would be an important proof of concept that could drive new drug development and a whole new strategy for helping to improve vision.”
Anti-Inflammatory Mechanisms of Disulfiram
Effects on Pyroptosis
What is Pyroptosis
Pyroptosis is a type of cell death, which was first discovered in 1992 and defined in 2001 (Zychlinsky et al., 1992; Cookson and Brennan, 2001), and many signaling pathways are involved in pyroptosis (Fink and Cookson, 2005). Change in cellular morphology is the formation of cell membrane holes, resulting in cell swelling and rupture, then the cell contents such as inflammatory factors are released to stimulate inflammatory responses (Jorgensen and Miao, 2015; Galluzzi et al., 2018). Pyroptosis can be classified into canonical inflammation pathway mediated by caspase-1 and non-canonical inflammation pathway mediated by caspase-4/5/11 (Jorgensen and Miao, 2015). Many inflammasomes are involved in pyroptosis, such as nod-like receptor protein 3 (NLRP3), absent in melanoma 2 (AIM2), NLRP1 and so on (Vande Walle and Lamkanfi, 2016). These inflammasomes can activate caspases that cut gasdermins into C-terminal and N-terminal gasdermins. The N-terminal gasdermins can perforate the cell membrane to cause cell swelling and rupture, so that release of inflammatory factors such as IL-1β and IL-18 results in inflammation (Ding et al., 2016), and completion of the pyroptosis process.
The Direct Effect of Disulfiram on Gasdermin D (GSDMD)
GSDMD is a member of the gasdermin protein family and plays an important role in the pyroptosis process. After being cleaved to N-terminal and C-terminal GSDMD by caspase-1/4/5/11, the N-terminal fragment can be transferred to the plasma membrane and form a membrane pore (Sborgi et al., 2016). In turn, IL-1β, IL-18 and other inflammatory mediators are released through this pore (Shi et al., 2015). Hu JJ et al. demonstrated in mouse experiment that DSF can inhibit the release of IL-1β without affecting other proteins such as caspase-1 and pro-IL-1β, thus confirming that DSF could inhibit pyroptosis by inhibiting GSDMD, and further confirming that DSF could covalently modify cys192 of GSDMD, rather than other gasdermins, inhibiting plasma membrane pore formation and the pyroptosis process (Hu et al., 2020). Furthermore, DSF can reduce the release of inflammatory mediators such as tumor necrosis factor (TNF) and IL-6 through the GSDMD pathway (Hu et al., 2020), thus reducing the inflammatory response to some extent. And it can be inferred that all pyroptosis pathways ending in GSDMD will be affected by DSF.
The Effect of Disulfiram on the Nod-Like Receptor Protein 3 Inflammasome and Indirect Effect on GSDMD
The NLRP3 inflammasome is one of the pyroptosis-associated inflammasomes leading to activation of caspase-1 which determines IL-1β and IL-18 maturation and release, contributes to pyroptosis (Jo et al., 2016). Deng et al. found that DSF could inhibit NLRP3, therefore inhibiting the release of IL-1β and the occurrence of cell pyroptosis in mouse J774A.1 and human THP-1 macrophage cell lines (Deng et al., 2020).
The activation of NLRP3 due to lysosomal destruction is also an important process during pyroptosis (Chen et al., 2015). Destabilization of the lysosomal membrane can lead to the release of cathepsin B, which activates the NLRP3 inflammasome (Newman et al., 2009; Jin and Flavell, 2010; Lamkanfi and Dixit, 2012), inducing the pyroptosis process. DSF can protect lysosomal membrane, reduce cathepsin B release therefore alleviating the inflammatory response (Deng et al., 2020).
The Dual Effects of Disulfiram on Reactive Oxygen Species
Reactive oxygen species (ROS), which play an important role in the development of many inflammatory diseases, are produced from the NADPH oxidase or the mitochondrial respiratory chain in the process of aerobic metabolism of organisms (Mittal et al., 2014; Blaser et al., 2016). A large number of literatures have confirmed that the production of ROS is one of the key elements of NLRP3 activation (Tschopp and Schroder, 2010; Bauernfeind et al., 2011), activation of NLRP3 can be blocked when using ROS inhibitors (Zhou et al., 2011). Recently, researchers have discovered that LPS/nigericin-induced mitochondrial ROS did not decrease after adding DSF, indicating that DSF can reduce intracellular ROS production by reducing the source of NADPH oxidase (Deng et al., 2020). Furthermore, researchers found that DSF can inhibit NLRP3-dependent IL-1β secretion (Deng et al., 2020), so it can be inferred that DSF can inhibit NLRP3 activity by reducing ROS production.
Disulfiram Inhibits Inflammation by Inhibiting Angiogenesis
Angiogenesis is an important stage in the inflammatory process, which is a key mechanism for leukocyte cells to enter the inflammation site through the vascular endothelium (Szekanecz and Koch, 2007). Angiogenic factors such as vascular endothelial growth factor (VEGF) and TNF-α can promote vessel formation (Leibovich et al., 1987; Marikovsky et al., 2003). DSF reduces TNF-α production and dose-dependently reduces the production of VEGF, thereby reducing angiogenesis and inflammation (Marikovsky et al., 2003). Another study has shown that DSF can reduce VEGF generation and inhibit angiogenesis in vivo by acting on the EGFR/Src/VEGF pathway, and this effect can be enhanced by the combination of copper (Li et al., 2015). Furthermore, DSF can cause ROS accumulation, induce intracellular oxidative stress, and cause endothelial cell growth arrest (Marikovsky et al., 2002), thus inhibiting angiogenesis.
Disulfiram and Inflammatory Disorders
The Therapeutic Effect of Disulfiram on Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disease of unknown etiology, including Crohn’s disease and Ulcerative Colitis (Moazzami et al., 2019), and has a certain risk of cancer (Bernstein et al., 2001). Clinically, abdominal pain and diarrhea are the main manifestations, which seriously affect the quality of life of patients (Sawczenko and Sandhu, 2003; Gupta et al., 2008). Oxygen free radicals play a role in tissue damage in the pathogenesis of colitis (Parks et al., 1983). Acetaldehyde dehydrogenase is responsible for the main source of ROS in colon (Sharon and Stenson, 1984), and the activity of ROS in tissues can be reflected in the level of molondialdehyde (MDA) when membrane lipids are damaged (Ohkawa et al., 1979). DSF can reduce MDA levels in the colonic tissues of rats with colitis (Bilsel et al., 2002). This may be due to the fact that DSF is the inhibitor of acetaldehyde dehydrogenase (Sawczenko and Sandhu, 2003; Gupta et al., 2008), however, the exact mechanism is still unclear.
Disulfiram for the Treatment of Inflammatory Liver Diseases
Non-alcoholic fatty liver disease (NAFLD) is a kind of disease which is characterized by excessive accumulation of lipids in liver cells, and is the most common chronic liver disease, which can be divided into simple steatosis and nonalcoholic steatohepatitis (NASH) (Haas et al., 2016; Younossi et al., 2016; Lau et al., 2017). NASH manifests itself as steatosis and inflammatory damage to the hepatocyte. NAFLD can develop into liver cirrhosis and even liver cancer. Rats receiving methionine and choline deficient (MCD) diet can develop NASH (Van Herck et al., 2017; Liu et al., 2018), with fat accumulation, oxidative stress, and inflammation and fibrosis in liver cells (Hebbard and George, 2011; Ibrahim et al., 2016; Lau et al., 2017). It manifests itself mainly as a reduction in serum cholesterol (CHO) and high-density lipoprotein (HDL) levels and an increase in alanine transaminase (ALT) and endoplasmic reticulum stress and an up-regulation of cytochrome P450 2E1 in mouse liver, which can produce superoxide anion radicals (Leung and Nieto, 2013). These manifestations can be inhibited by the treatment with the diethyldithiocarbamate (DDC), the DSF metabolites (Liu et al., 2018). What’s more, DDC treatment can improve the liver function damage caused by MCD diet (Liu et al., 2018). Besides, the use of tetraethylthiuram DSF (TDSF) in animals on the MCD diet resulted in a significant reduction in inflammatory cell infiltration in the liver (Schwartz et al., 2013).
Alcoholic liver disease is a chronic liver disease caused by long-term heavy alcohol consumption, which can cause alcoholic hepatitis (Hines and Wheeler, 2004; Jerrells et al., 2007; Gao et al., 2019), and may develop into liver cancer (Altamirano and Bataller, 2011). Acetaldehyde levels increased in ALDH2 (-) mice when exposed to alcohol, and hepatitis was attenuated in ALDH2 (-) mice compared to wild-type mice (Hines and Wheeler, 2004; Jerrells et al., 2007; Gao et al., 2019). It can be inferred that DSF, as an ALDH inhibitor, can reduce hepatitis caused by alcohol use.
Mice on MCD diet developed liver fibrosis and collagen deposition can be observed around the central lobular vein. By reducing the aggregation of hepatic stellate cells (HSC) and myofibroblasts, DDC significantly reduced liver fibrosis induced by MCD diet (Hines and Wheeler, 2004; Jerrells et al., 2007; Gao et al., 2019).
Disulfiram for Inflammatory Kidney Diseases
Renal fibrosis is the pathological response of the kidney to a variety of pathogenic factors such as inflammation and ischemia. When the kidney is stimulated by injury, the inflammatory pathway is activated and further activates pro-fibrotic cells (Black et al., 2019). NLRP3 plays an important role in the activation of renal fibrosis (Granata et al., 2015; Zhang and Wang, 2019). In the mouse model with NLRP3 knockout diabetic nephropathy, renal inflammation and fibrosis can be partially suppressed, suggesting that the pro-inflammatory effect of the NLRP3 inflammasome may promote renal fibrosis (Wu et al., 2018). Unilateral ureteral obstruction (UUO) is an experimental model of kidney injury and can cause renal fibrosis. After using DSF on UUO rats, the expression of IL-1β, IL-6, IL-18 and TNF-α in the peripheral blood and kidney tissues of the rats decrease significantly, and the degree of reduction was negatively correlated with the drug dose. What’s more, the use of DSF reduces the expression of GSDMD, and DSF could downregulate the level of α-SMA and upregulate the level of E-cadherin in renal tissues. These findings indicate that DSF can ameliorate renal fibrosis of UUO rats by inhibiting pyroptosis and other pathways (Zhang et al., 2021). Other studies have shown that DSF can reduce cisplatin-induced acute renal toxicity in rats by decreasing oxidative stress and inflammation (Khairnar et al., 2020).
Disulfiram for Sepsis
Sepsis is a severe systemic inflammatory response following infection (Heumann et al., 1998), with a mortality rate of about 25% when no complication occurs or 80% when accompanied with multiple organ failure (Galley, 2011). Lipopolysaccharide (LPS) in the cell wall of Gram-negative bacteria is one of the stimulating factors that can cause sepsis (Heumann et al., 1998). LPS can induce sepsis in mice, while caspase-11 (-) mice were not induced to sepsis, suggesting that the non-canonical inflammasome pathway dominates LPS-induced sepsis (Kayagaki et al., 2011). Hu JJ at al. reported that DSF can prolong the survival time of LPS-induced sepsis mice. By the combination of copper, the therapeutic effect of DSF was further strengthened. These results suggest that DSF can inhibit sepsis induced by the non-canonical pyroptosis pathway (Hu et al., 2020).
Disulfiram for Type 2 Diabetes
Type 2 diabetes is one of the most common diseases in internal medicine and has a high incidence in middle-aged and elderly people. Its hyperglycemic characteristic can lead to a series of complications (Vijan, 2015; American Diabetes Association, 2020). The onset of type 2 diabetes is associated with decreased pancreatic beta cell function and insulin resistance (Nagai et al., 2009; Zheng et al., 2018). Studies have shown that the onset of type 2 diabetes is related to ROS: glycation and the consequent increase of ROS can inhibit the transcription of insulin genes in mouse β-cell–derived HIT-T15 cells (Matsuoka et al., 1997). Based on rat models of type 2 diabetes, researchers found that oral administration of DSF increased insulin levels, improved glucose tolerance and reduced blood glucose and cholesterol levels in diabetic rats (Nagai et al., 2009). Furthermore, researchers found that DSF and its derivatives can covalently bind to the C128 site to inhibit fructose-1,6-bisphosphatase (FBPase), an important rate-limiting enzyme in the process of gluconeogenesis, thereby reducing blood glucose output (Huang et al., 2020). However, more in-depth research is needed on the treatment of DSF for diabetes.
Disulfiram for Uveitis
Uveitis is considered an inflammatory disease that occurs in the uvea, the retina, the blood vessels of the retina, and the vitreous, and can cause blindness (Nussenblatt, 1990). The main pathophysiological characteristics are infiltration of inflammatory cells and accumulation of inflammatory factors in aqueous humor (Mo et al., 1999). DSF can dose-dependently reduce inflammatory cells infiltration and protein concentration in aqueous humor of LPS-induced rat model of uveitis, and decrease the levels of inflammatory factors such as NO, TNF-α and PGE2 in aqueous humor (Kanai et al., 2010; Kanai et al., 2011). However, its mechanism needs to be further elucidated.
Disulfiram for the Inflammatory Response of Chondrocytes
Osteoarthritis is a chronic bone and joint disease that is more likely to occur in middle-aged and elderly people (Lespasio et al., 2017). In severe cases, it can cause joint pain and interfere with normal movement (Huang et al., 2015; Lespasio et al., 2017). Degenerative changes in articular cartilage can result in osteoarthritis (Goldring and Berenbaum, 2015). In C28/I2 cells (human chondrocytes), LPS and ATP can induce inflammation and pyroptosis (Li et al., 2021). Co-treatment with disulfiram and glycyrrhizic acid promote the proliferation and alleviate pyroptosis in LPS and ATP stimulated C28/I2 chondrocytes, and can reduce the production of ROS. DSF, when used at high concentrations, shows little effect on cell proliferation and pyroptosis (Li et al., 2021). This may provide new ideas for the treatment of osteoarthritis.
reference link :https://www.frontiersin.org/articles/10.3389/fphar.2021.795078/full
More information: Michael Telias et al, Retinoic acid inhibitors mitigate vision loss in a mouse model of retinal degeneration, Science Advances (2022). DOI: 10.1126/sciadv.abm4643. www.science.org/doi/10.1126/sciadv.abm4643