SULT2A1

Over 12 million people worldwide suffer from a chronic infection with the hepatitis D virus. This most severe viral liver disease is associated with a high risk of dying from liver cirrhosis and liver cancer. It is caused by the hepatitis D virus (HDV), which uses the surface proteins of the hepatitis B virus (HBV) as a vehicle to specifically enter liver cells via a protein in the cell membrane -- the bile salt transporter protein NTCP. Over 12 million people worldwide suffer from a chronic infection with the hepatitis D virus. This most severe viral liver disease is associated with a high risk of dying from liver cirrhosis and liver cancer. It is caused by the hepatitis D virus (HDV), which uses the surface proteins of the hepatitis B virus (HBV) as a vehicle to specifically enter liver cells via a protein in the cell membrane -- the bile salt transporter protein NTCP. This cell entry can be prevented by the active agent bulevirtide, which is approved as a drug under the name Hepcludex. An international research team has now succeeded in deciphering the molecular structure of bulevirtide in complex with the HBV/HDV receptor NTCP at the molecular level. The research results published in the journal Nature Communications pave the way for more targeted and effective treatments for millions of people chronically infected with HBV/HDV. The entry inhibitor bulevirtide is the first and currently only approved drug (under the drug name Hepcludex) for the treatment of chronic infections with the hepatitis D virus. The active agent effectively inhibits the replication of hepatitis D viruses and leads to a significant improvement in liver function. However, the exact mechanism by which bulevirtide interacts with the virus entry receptor on the surface of the liver cells -- the bile salt transporter protein NTCP (short for: sodium taurocholate cotransporting polypeptide) -- and thereby inhibits the entry of the viruses into the cells was previously unknown. In order to understand the molecular interaction of bulevirtide and NTCP at the molecular level, the researchers first generated an antibody fragment that specifically recognises the NTCP-bulevirtide complex and makes it accessible for analysis when bound to nanoparticles. This complex was then analysed using cryo-electron microscopy, which allowed to visualise structural details with atomic resolution. The research results represent a milestone in understanding both the interaction of HBV and HDV with their cellular entry receptor NTCP and the mechanism of cell receptor blockade by bulevirtide. How bulevirtide blocks the cell entry receptor NTCP The analysis showed that bulevirtide forms three functional domains in the interaction with the HBV/HDV receptor NTCP: a myristoyl group that interacts with the cell membrane on the outside of the cell; an essential core sequence ('plug') that fits precisely into the bile salt transport tunnel of the NTCP like the bit of a key into a lock; and an amino acid chain that stretches across the extracellular surface of the receptor, enclosing it like a brace. "The formation of a 'plug' in the transport tunnel and the associated inactivation of the bile salt transporter is so far unique among all known virus-receptor complexes. This structure explains why the physiological function of the NTCP is inhibited when patients are treated with bulevirtide," says Prof Stephan Urban, DZIF Professor of Translational Virology and Deputy Coordinator of the DZIF research area Hepatitis, in whose laboratory at Heidelberg University the active agent bulevirtide was developed. "Thanks to the structural details of the interaction with bulevirtide, we have also gained insights that enable the development of smaller active agents -- so-called peptidomimetics -- with improved pharmacological properties. Our structural analysis also lays the foundation for the development of drugs that are not only based on peptides and possibly enable oral administration," adds the co-author of the study, Prof Joachim Geyer from the Institute of Pharmacology and Toxicology at Justus Liebig University Giessen. Evolutionary adaptation of hepatitis B viruses to host species The structural analysis also helped to decode an important factor in the species specificity of hepatitis B and D viruses. According to the findings of the analysis, the amino acid at position 158 of the NTCP amino acid chain plays an essential role in virus-receptor interaction. A change in the amino acid at this position prevents the binding of HBV/HDV. This explains why certain Old World monkeys, such as macaques, cannot be infected by HBV/HDV. "Our findings enable a deeper understanding of the evolutionary adaptation of human and animal hepatitis B viruses to their hosts and also provide an important molecular basis for the development of new and targeted drugs," adds co-author Prof Dieter Glebe, DZIF scientist at the Institute of Medical Virology at Justus Liebig University Giessen. "Our research results are an important step in the fight against hepatitis D and B. By understanding the structure of bulevirtide and its binding to NTCP, we can potentially develop more targeted and effective treatments for millions of people chronically infected with HBV/HDV," says Prof. Kaspar Locher, last author of the publication and head of the structural biology team at ETH Zurich, summarising the study results.

Bile acids long have been known to play a role in human metabolism. Synthesized from cholesterol in the liver, bile acids are involved in digestive processes, particularly in absorbing fat. They also are modified extensively by bacteria, which greatly expand the types of bile acids found in the host. For most of a century, scientists believed that was the end of the bile-acid story. Recent technological advances, however, have led to a greater understanding of the origins of bile acids as well as their chemical relationships to the organisms in the gut microbiome and their host. Bile acids long have been known to play a role in human metabolism. Synthesized from cholesterol in the liver, bile acids are involved in digestive processes, particularly in absorbing fat. They also are modified extensively by bacteria, which greatly expand the types of bile acids found in the host. For most of a century, scientists believed that was the end of the bile-acid story. Recent technological advances, however, have led to a greater understanding of the origins of bile acids as well as their chemical relationships to the organisms in the gut microbiome and their host. Deploying some of these technologies, a team led by Penn State researchers has uncovered the mechanism by which bacteria generate a wide variety of new bile acid species, the functions of which are not yet clear. The researchers, who published their results recently in Nature, identified a new role for an old bacterial enzyme, known as bile salt hydrolase, or BSH. The enzyme modifies human- and mouse-generated bile acids and changes their configurations by, for example, adding amino acids, leading to new molecules known as bacteria bile acid amidates, or BBAAs. The team also showed, for the first time, that these BBAAs are made in humans at birth, coinciding with the establishment of the gut microbiome in newborns. "The influence of bile acids on health and disease is well established," said corresponding author Andrew Patterson, professor of molecular toxicology and the John T. and Paige S. Smith Professor in Penn State's College of Agricultural Sciences. "But now we're finding that they can serve as signaling molecules between us and our microbial counterparts. It's like a communication network between us and microbes, with bile acids being the messenger." Patterson, who also holds an appointment as professor of biochemistry and molecular biology in the Eberly College of Science, explained that the hundreds -- and perhaps thousands -- of new bile acid species created by BSH-producing bacteria may have far-reaching signaling properties. "Researchers have reported finding bile acids in the brain, skin and other tissues," he said. "This suggests that they probably have a broader role beyond just helping us consume fat. Discovering what this role could be is a question we're still trying to answer, and that's what's really exciting about this research." Although the researchers are unsure about the long-term implications of their study, they pointed out that BSH and bile acids have been linked with many health conditions, such as inflammatory bowel disease, some cancers and obesity. Understanding their role eventually could lead to therapies, they said. "There are many diseases associated with abnormal lipid or bile acid metabolism -- if you can't absorb fat properly, you're going to have problems," Patterson said. "Teasing out the functions of these new bile acid species opens a lot of new doors to explore." To test their hypothesis that bile salt hydrolase is involved in the creation of bile acids, the researchers took a multipronged approach. In some experiments, they used an inhibitor to block the bacterial activity of BSH, and in others, they modified bacteria to remove the gene that encodes for BSH. In the absence of BSH, there was no production of BBAAs, the team found. Working with the Children's Hospital of Philadelphia and the University of Pennsylvania, the researchers also tested stool samples of infants from birth to 12 months old. They found that a rise in bile acids, including BBAAs, coincided with the colonization of BSH-producing bacteria in the infants' guts. It is the first time scientists have connected BBAA production with BSH-expressing bacteria during human infant development, according to the researchers. The results of this study reinforce, and are reinforced by, a parallel study that was conducted at Michigan State University and published in the same issue of Nature. Although the research teams did not work together directly -- and approached the topic from different angles -- they shared information and collaborated to publish their studies simultaneously, Patterson explained. "Publishing these papers at the same time was a great example of land-grant collaboration," he said. "Having two independent labs come up with the same answer really bolsters the significance of the findings, which in some ways upend 100 years of thinking in the field." Robert Quinn, whose lab published the MSU study, noted that his team showed that the BSH enzyme produced bile acids, while the Patterson lab did the reverse, identifying the enzyme and inhibiting it, which revealed that these molecules went away. "It was perfectly complementary," said Quinn, assistant professor in MSU's Department of Biochemistry and Molecular Biology. Bipin Rimal, co-first author of the Penn State paper, echoed the sentiment. "These highly interdisciplinary collaborations at Penn State, Michigan State and places across the country helped transform a simple idea into something that reshapes our understanding of the role of BSH," said Rimal, who received his doctorate in 2023 while working in Patterson's lab. Patterson credited talented graduate students, a diverse interdisciplinary team cooperating across campus and coast-to-coast, and the availability of technologies such as mass spectrometry and nuclear magnetic resonance platforms for making his lab's discoveries possible. Penn State study co-first author Stephanie Collins, a doctoral alumna of the Patterson Lab, agreed. "Without the technological capabilities and having the right mix of people here at the right time, we could not have accomplished what we did," she said. Also part of the research team from Penn State were Megan Granda, Fuhua Hao and John Vanden Heuvel, Department of Veterinary and Biomedical Sciences; Nushrat Hoque, Department of Chemistry; Imhoi Koo, Huck Institutes of the Life Sciences; Erin Reilly, Min Soo Kim, Jordan E. Bisanz and Emily Weinert, Department of Biochemistry and Molecular Biology; Devendra Paudel and Vishal Singh, Department of Nutritional Sciences; and Dhimant Desai and Shantu Amin, Department of Pharmacology. Other researchers on the paper represented Children's Hospital of Philadelphia, East Carolina University, the University of Michigan, the University of California San Diego, the National Cancer Institute and the University of Pennsylvania. The National Institutes of Health, the Rosalind E. Franklin Science Achievement Graduate Fellowship program in Penn State's Eberly College of Science, the Huck Institutes of the Life Sciences at Penn State, the American Beverage Foundation for a Healthy America, the Crohn's and Colitis Foundation, the American Heart Association postdoctoral fellowship program, the Pennsylvania Department of Health, the U.S. Department of Agriculture's National Institute of Food and Agriculture, and Penn State supported this work.

Product launched in India is exceeding sales expectations and is slated for introduction into 30 other countries through a distribution partnership with Cosmofix & San Pellegrino. Added Dr. Ready’s, a 2.99B revenue yearly company, to our liciensee listClinical trial results demonstrating efficacy in hair growth expected Q4 2023—data to support upcoming launch of product in the U.S. and other major markets JUPITER, FL, Oct. 25, 2023 (GLOBE NEWSWIRE) -- Safety Shot Inc. (Nasdaq: SHOT) today announced the U.S. Patent and Trademark Office has issued Patent No. US 11,766,392 for JW-700, a topical treatment designed to improve minoxidil’s efficacy, marking a significant milestone in the development of treatments for androgenetic alopecia, or hair loss. This is the second U.S. patent issued for JW-700 this year and adds to the growing global IP estate for Safety Shots Legacy products. Minoxidil is a widely used for the treatment of hair loss sold under brand names including Rogaine® and Hair Regrowth for Men®. JW-700 is clinically shown to increase the sulfotransferase enzymes needed for minoxidil to work by up to 7-X over a two-week period. This issued patent discloses composition and methods for inducing the expression of sulfotransferases in hair follicles and expands patent protection on JW-700’s new formulation. An ongoing clinical study of JW-700 in India was recently completed and is now in the final stages of analysis. JW-700 has been licensed to Taisho, a $2.6 billion revenue company, in Japan, and to Sanpellegrino Cosmetics and Cosmofix Technovation in India, where it is marketed under the brand name MinoxiBoost, as well as 30 other countries in Asia and the Middle East including the Philippines, Malaysia, Kuwait, Morocco, and Saudi Arabia. In India product is marketed by leading companies, , Glenmark, Eris-Oaknet, Cipla and now Dr Reddys (November 2023 launch) “This new, broader U.S. patent, encompassing new formulations, is a crucial component of Safety Shot’s ongoing pursuit of the $1.5 billion minoxidil hair loss market. Following its launch in India in July of 2023, sales of MinoxiBoost have exceeded all expectations and we expect our distribution partners will continue to deliver robust sales,” stated Safety Shot’s CEO Brian John. “The recently completed clinical trial is a vital next step to support a full launch of JW-700 in the U.S. and other key markets. We look forward to reporting data in the coming weeks as well as the launch of this incredible product in the USA.” Safety Shot is focused on its functional beverage, Safety Shot, the first patented beverage on Earth that helps people feel better faster by reducing blood alcohol content and boosting clarity. The Company is consolidating the rest of its legacy healthcare and wellness assets, including JW-700, into a subsidiary which it intends to list publicly as a separately traded entity for the benefit of Safety Shot and its shareholders. About Safety Shot Safety Shot, a wellness and functional beverage company, is set to launch Safety Shot, the first patented beverage on Earth that helps people feel better faster by reducing blood alcohol content and boosting clarity. The Company plans to spin off legacy assets from its Jupiter Wellness business to unlock value for shareholders. Interested investors and shareholders are encouraged to sign up for press releases and industry updates by registering for Email Alerts at https://jupiterwellness.com/email-alerts/ and by following Jupiter Wellness on X (formerly known as Twitter) and LinkedIn. Forward Looking Statements This communication contains forward-looking statements regarding Safety Shot, including, the anticipated timing of studies and the results and benefits thereof. You can generally identify forward-looking statements by the use of forward-looking terminology such as “anticipate,” “believe,” “continue,” “could,” “estimate,” “expect,” “explore,” “evaluate,” “intend,” “may,” “might,” “plan,” “potential,” “predict,” “project,” “seek,” “should,” or “will,” or the negative thereof or other variations thereon or comparable terminology. These forward-looking statements are based on each of the Company’s current plans, objectives, estimates, expectations, and intentions and inherently involve significant risks and uncertainties, many of which are beyond Safety Shot’s control. Actual results and the timing of events could differ materially from those anticipated in such forward-looking statements as a result of these risks and uncertainties and other risks and uncertainties affecting Safety Shot and, including those described from time to time under the caption “Risk Factors” and elsewhere in Safety Shot’s Securities and Exchange Commission (SEC) filings and reports, including Safety Shot’s Annual Report on Form 10-K for the year ended December 31, 2023 and future filings and reports by Safety Shot. Moreover, other risks and uncertainties of which the combined company is not currently aware may also affect each of the companies’ forward-looking statements and may cause actual results and the timing of events to differ materially from those anticipated. Investors are cautioned that forward-looking statements are not guarantees of future performance. The forward-looking statements made in this communication are made only as of the date hereof or as of the dates indicated in the forward-looking statements and reflect the views stated therein with respect to future events at such dates, even if they are subsequently made available by Safety Shot on its website or otherwise. Safety Shot undertakes no obligation to update or supplement any forward-looking statements to reflect actual results, new information, future events, changes in its expectations or other circumstances that exist after the date as of which the forward-looking statements were made. Safety Shot Media Contact: Phone: 561-244-7100Email: investors@safetyshotholdings.com ###