O’Shaughnessy’s • Winter 2015/16 —15— Appendino’s Advice to Cannabinoid Researchers: Consider ‘New Targets, Chemistry, and Plant Sources’ By Ryan Lee and O’S News Service “Natural Selection Works Like a Tinkerer...” The International Cannabinoid Research monoterpenes (limonene, pinene, linalool, Society held its 24th annual meeting at a terpinolene, et al). lakeside hotel in Baveno, Italy, in June The 15-carbon sesquiterpenes such as 2014. ICRS members are mainly —but beta-caryophyllene, differ from the mono- not exclusively— university-connected terpenes by the incorporation of an extra biochemists and pharmacologists investi- isoprene unit. (β is the Greek letter beta.) gating how things work at the sub-cellular level. When Cannabis is dried, stored Baveno is a resort town on big, beauti- for periods of time, or made into ful Lake Maggioro, with the Alps visible extracts, the monoterpenes are gen- to the north. There were four days of talks erally first to evaporate. The sesqui- describing recent studies, and sessions at which investigators answered questions terpenes like β-caryophyllene are about their findings as summarized on more likely to remain. posters. When the ICRS was founded in 1990, Monoterpenes are more volatile —they its original name was “International Can- evaporate at lower temperatures— so when nabis Research Society.” In 1995 —after Cannabis is dried, or stored for periods of the body’s own cannabinoid receptor sys- time, or made into extracts, the monoter- tem had been discovered and elucidated by penes are generally first to evaporate. The ICRS members— the group changed the sesquiterpenes like β-caryophyllene are C-word in its name to “Cannabinoid.” As more likely to remain. pharmacologist Dale Deutsch explained in β-caryophyllene seems like the Cannabis plant’s own perfect key for nature’s CB2 1998, “The field is moving away from the Leaves that resemble Cannabis sativa are (top row, left to right): Acer japonicus, Ac- lock. Plants use β-caryophyllene to defend plant.” onitum vulparia, Geranium pratense. Bottom row, left to right: Hibiscus cannabinum, themselves against predators. Some spe- The 2014 ICRS meeting marked the re- Vitex agnus-castus, Cannabis sativa. Graphic from “Plantes interdites. Une histoire cies up-regulate specific terpenes when turn of the plant to the forefront of the field. des plantes politiquement incorrectes,” by Jean-Michel Groult. Appendino quoted attacked by herbivores to render the plant Neurologist Ethan Russo was serving as the French scientist Francois Jacob in connection with this slide:: “Natural selection less palatable to the attacking insect. ICRS president (the job is held for a year), works like a tinkerer who does not know exactly what he is going to produce, but In a beautiful demonstration of the web and he invited the Italian natural product uses whatever he finds around him to produce some kind of workable object. None of that Mother Nature has created, these same chemist Giovanni Appendino to give the the material at the tinkerer’s disposal has a precise and definite function. Each can terpenes have been shown to recruit para- featured talk at the meeting in Baveno. be used in different ways. Novelty comes from previously unseen association of old sitic bugs that themselves attack the herbi- Appendino, a professor at the Università material. To create is to recombine.” del Piemonte Orientale, noted proudly that vores that are eating the plant! he is from Carmagnola, a northern Italian reminded his ICRS audience. also impede legitimate scientific research. town renowned for its fiber hemp variety In the course of screening more than 200 After two years of bureaucratic red tape, The drive to breed high-yielding of the same name. varieties of fiber hemp, Appendino and Appendino was only able to obtain a small varieties of corn for intensive Appendino first published research in the colleagues have found significant quanti- vial of extract from the plant. Being unable cannabinoid field in 2002, when he was co- ties of obscure compounds whose medical to obtain seeds themselves has limited his commercial agriculture sacri- author of a paper on “Noladin ether —a pu- potential he considers “worthy of investi- ability to investigate the biosynthetic path- ficed the ability of the plant to tative endocannabinoid.” (The lead authors gation.” ways by which Helichrysum produces can- produce β-caryophyllene . were Raphael Mechoulam and Vincenzo nabinoids. DiMarzo.) But Appendino’s “relationship Cannabinoids are not unique to Appendino discovered that cannabinoid- Appendino recounted how the wild, an- with cannabis as fiber hemp” goes much like compounds are made by plants “apart Cannabis —they have been found cestral relative of corn, teosinte, grown by further back: “My grandfather was grow- from the normal cannabinoid biosynthetic the Mayan and Incan farmers in pre-Euro- ing it and the odor of hemp retting tanks in other plants. route. There is a new pathway that starts pean Central and South America, produced was filling the air around Carmagnola dur- from an aromatic acid.” Referred to as the significant amounts of β-caryophyllene ing the Fall.” He touched briefly on canniprene, the “Helichrysum cannabinoids,” these com- before modern breeders selected towards cannflavins, cannabinoid esters, and “ses- pounds also have been detected in liver- high yielding corn with an increased sugar By defining cannabinoids as qui-CBG,” which Appendino’s group iso- wort. content. The drive to breed high-yielding lated from a fiber hemp variety. drugs that work at the CB1 and Helichrysum is used in African ethno- varieties of corn for intensive commer- Appendino has encountered a hemp vari- pharmacology, Appendino explains, “like cial agriculture sacrificed the ability of the CB2 receptors, researchers may ety containing two percent canniprene —a hemp, to make fumes in ritual ceremonies” plant to produce β-caryophyllene . Cannabis be overlooking beneficial com- compound he called “the version and that a “psychotropic effect... similar to That β-caryophyllene binds specifically of resveratrol” (a beneficial compound cannabinoids,” might ensue. to the CB2 receptor (which is found main- pounds in Cannabis that work by present in red grapes). other mechanisms. ly outside the central nervous system) was From others varieties Beta-caryophyllene reported by Jürg Gertsch at the 2007 ICRS he isolated the prenyl- Terpenoids, the largest class of naturally Researchers have focused almost ex- meeting. ated version of cannab- occurring compounds on the planet, are clusively on THC, CBD, CBC (canna- igerol —meaning CBG the chemicals that give plants their unique bichromene) and CBG (cannabigerol, pre- The CB2 receptor attached to a prenyl group (illustration at smells and flavors. Found in high concen- cursor to the other three), Appendino said, The CB2 receptor has yet to be success- left). There is no reason, Appendino said, trations in many culinary herbs and spices, while not investigating the therapeutic po- fully exploited by the pharmaceutical that marijuana should not also produce the terpenes not only provide flavor and scent, tential of related molecules present in Can- industry, Appendino said. “If drug dis- prenylated version of THC —which would they are also important signaling chemi- nabis —and other plants as well. covery is a sea, then CB2 is a rock that is have distinct biological activity. cals that plants use to communicate with Similarly, by defining cannabinoids as surrounded by shipwrecked-projects,” he insects. drugs that work at the CB1 and CB2 recep- Cannabinoids not unique to Cannabis commented poetically. Terpenes are synthesized by the plant tors, researchers may be overlooking ben- Cannabinoids are not unique to cannabis Pharmaceutical companies have spent from five-carbon isoprene units, two of eficial compounds in Cannabis that work —they have been found in other plants. which come together in specialized cel- continued on next page by other mechanisms. “Nature has varied Appendino reported that a large amount of lular compartments to form the 10-carbon on the cannabinoid structure,” Appendino CBG and its carboxylic precursor had been isolated from a specific He- lichrysum variety found only in South Africa. Studying how Helichrysum makes “non-cannabis” CBG and its related compounds has been difficult for - Ap pendino and his colleagues, alpha-pinene beta-pinene limonene βeta caryophylllene because strict South African Terpenoids are categorized in terms of how many 5-carbon bio-piracy laws prohibit the units they contain. Three molecules at left are monoter- collection and export of na- penes —each contains 10 carbon atoms. Larger molecule tive species or their seeds. at right, β-caryophyllene, is a sesquiterpene with 15 carbon Helichrysum umbraculigerum, a daisy na- These laws, designed to atoms. Because β-caryophyllene is heavier than the mono- tive to South Africa, produces cannabigerol prevent foreign corporate terpenes, it evaporates less readily and is often present in Teosinte, the ancestor of corn was tiny but (CBG). It was identified by Ferdinand Bohl- exploitation of the country’s relatively large amounts in dried Cannabis. (But not all rich in beta-caryophyllene. The cob in this mann and Evelynn Hoffmann in 1978. unique genetic resources, Cannabis produces large amounts of β-caryophyllene.) photo is two inches tall. —16—O’Shaughnessy’s • Winter 2015/16 ICRS from previous page large sums investigating proprietary synthetic CB2- selective compounds that end up showing little clinical effi- cacy. “But β-caryophyllene is a special lottery ticket,” said Appendino. β-caryophyllene is known to be anti-inflammatory and easy on the stomach lining. A special lottery ticket, indeed! So grind some black pepper on your next salad, and order Cannabigerol (CBG, top left) is the compound in Cannabis from which other plant cannabi- those Echinacea and mari- noids are synthesized. Molecule at bottom left is sesqui-CBG, which has been identified in fi- gold seeds now —they all ber hemp It consists of CBG plus a five-carbon pentyl tail (at right in illustration). Appendino contain β-caryophyllene. posits the existence of Sesqui-THC, a plant compound consisting of THC plus a pentyl group. CBD (top left) and Ferruginene C (top right) have Appendino described how similar molecular structures. Ferruginene C is pro- the β-caryophyllene mol- Drugs like VCE-003, made by adding cept of the endocannabinoid system. In duced by Rhododendron ferrugineum, an Alpine ever- ecule interacts with the CB2 side chains to naturally occurring mol- addition to CB1 and CB2, the biological green shrub (photo at right). Photo at left is of a CBD- receptor. It’s an unusual ecules, are known as “semi-synthetics.” targets of the expanded-definition canna- rich variety called “ACDC,” grown and photographed physical relationship for Hydrocodone and bupenorphine, which binoids include the GPR55 receptor; TRPs by Lawrence Ringo. cannabinoid-type agonists. have replaced codeine and morphine and (pronounced “trips”), which are tiny ion β-caryophyllene does not VCE-003, which outperforms CBG in ac- most opioid analgesics now sold in the channels with gates that open and close to look like any other molecule that binds to tivating PPAR receptors. VCE has shown U.S., are well-known semi-synthetics. transmit signals; and transcription factors the cannabinoid receptors. efficacy in studies using mouse models of Appendino’s expanded definition of can- in the mitochondria that switch genes on Extracts from plants high in Multiple Sclerosis and Encephalomyelitis. nabinoid drugs involves an expanded con- and off. β-caryophyllene have shown some anal- gesic effect in clinical trials. “Maybe the interaction of β-caryophyllene with CB2 is an echo of an ancient dialog between plants and insects,” Appendino said.

Expanded-Definition Cannabinoids CBG (top) and VCE-003 Just as natural selection tinkers with com- pounds, so do scientists, hoping to find a useful modification that evolutionary pres- 8PN (8-Prenylnaringenin, left), a flavonoid prevalent in hops, is the most potent estro- sure hasn’t induced nature to come up genic compound found in plants. Its effects are similar to, but weaker than the hormone with. Research is underway into some of estradiol (center). Pointing out the similar structure of flavonoids found in Cannabis, Ap- the unorthodox cannabinoids Appendino pendino asked, “Could Cannflavin be the estrogenic principle ofCannabis? ” Chemist Matt Giese adds, “These type of flavonoids can form isomers, where the methoxy (OMe) and discussed. ICRS coverage continued on next page. For example, a Spanish biotech compa- hydroxy(OH) groups have switched positions. This can greatly affect binding and function- ny called VivaCell has developed a drug, ality, which is why A and B have such different activities.” The Flavonoids Unique to Cannabis Flavonoids are compounds produced by would counter the activity of an inflamma- many plants that influence the color of tory mediator, prostaglandin E2 (PGE2), flowers, among other things. Flavonoids present in synovial cells cultured from the are defined by a 15-carbon backbone that knee joints of patients undergoing surgery includes two phenyl groups. Like terpe- for rheumatoid arthritis. noids, they are “secondary metabolites,” Barrett and her co-workers found that a advantageous to the plant (attracting a pol- cannabinoid-free alcohol extract of Can- linator, inhibiting growth of a mold, etc.) nabis was inhibiting the release of these but not “primary” components like the pro- inflammatory prostaglandins from the Cannflavin A (structure 1) and cannflavin B (structure 3) were isolated from Cannabis teins, lipids, and carboydrates needed for cultured cells. To determine which compo- in the 1980s by Barrett et al. These are prenylated flavones—compounds that have a pre- life itself. nent of the extract was having the anti-in- nyl group (3-methyl-but-2-en-1-yl) attached to their flavonoid backbone. Cannflavin B is In the 1980s, Dr. Marilyn Barrett identi- flammatory effect, they divided the extract different from A, lacking the five carbon alkyl unit at C-4. (Structure 2 was included for fied two diprenylated flavonoids in Canna- into fractions (using preparative thin layer purposes of structure identification; it is not a compound found inCannabis.) bis which were previously unknown. She chromatography) and measured the activ- (Fairbairn and Pickens) at the School of named them “Cannflavin” A and B. ity of each fraction in the cell culture as- Barrett first published her account of iso- Pharmacy used a model of catalepsy in In 2013 Mahmoud ElSohly and col- say. The most active fraction, in turn, was lating Cannflavin in Biochemical Pharma- mice to measure the psychoactive proper- leagues at the University of Mississippi divided into fractions and its most active cology, June 1985. Details of the structure ties of the cannabinoids, particularly THC. identified a third, Cannflavin C. fraction selected, and the process repeated elucidation were published in a second pa- They were extracting the cannabinoids As noted by Giovanni Appendino at the until a pure compound was isolated. per in Experientia 42, April 1986. from dried plant material using petroleum 2014 ICRS meeting, Cannflavins are now “Once you get down to a pure compound,” Although Barrett’s team found Cannfla- spirit until the remaining plant material being studied for anti-inflammatory activ- Barrett explains, “then you can work on vin to be 30 times more potent than aspirin was cannabinoid-free. The spent plant ma- ity, and hemp cultivars with unusually high identifying the structure of that compound. as an anti-inflammatory in the cell culture terial was then extracted with alcohol. cannflavin content (c. 2%) are being grown We used mass spectrometry (MS) to mea- assay, it was 18 times weaker than Indo- Fairbairn and Pickens determined that in Italy. sure the molecular weight along with pro- methacin —which is perhaps why no effort this alcoholic extract of Cannabis, which We sought some background info from ton- and carbon- nuclear magnetic spec- was made to develop Cannflavin as a drug. was free of cannabinoids, had the ability to Barrett, who is based in Mill Valley. troscopy to get a picture of the structure. “Especially interesting, from a scientific counteract the cataleptic activity of THC in Barrett describes her discovery as “clas- Ultraviolet spectroscopy confirmed we point of view,” Barrett notes, “might be mice. They suspected that the inhibition of sic pharmacognosy.” She was a PhD stu- were working with a flavonoid, belonging that the Cannabis plant contains substanc- prostaglandins was important to this effect, dent at the School of Pharmacy, University to the class of flavones es that both cause and reduce a cataleptic and in confirmation of this idea, inhibitors of London, looking for compounds that Previous work by Barrett’s colleagues effect in mice.” This finding was duplicat- of cyclooxygenase also demonstrated this ed in the synovial cell assay, in which the activity in mice. It was this work that led to cannabinoids stimulated the production of Barrett’s search for an anti-inflammatory PGE2 and Cannflavin had an inhibitory ef- agent in the alcoholic extract. fect. —O’S News Service

Paper by Marilyn Barrett and colleagues in “Biochemical Pharmacology,” June 1985 de- scribed the isolation of Cannflavin, the first in a new group of diprenylated flavones.

Copyright 2015 by O’Shaughnessy’s. All rights reserved. Address reprint requests to [email protected] O’Shaughnessy’s • Winter 2015/16 —17—

ICRS from previous page The 2014 ICRS meeting was attended by 328 people —a record. Major pharmaceu- tical companies that used to send scientists to present papers and monitor the latest research—Abbott, Allergan, AstraZeneca, Bayer, Bristol-Myers Squibb, GlaxoSmith- Kline, Merck, Pfizer, Eli Lilly and Sanofi- Aventis—were not represented in Baveno. At least for now, Big PhRMA seems to have surrendered to the plant itself as a source of cannabinoid drugs. The one exception was Hoffman-La- Dale Deutsch reported that the molecules Roche. Researchers employed by the Ba- that transport anandamide and 2-AG from the receptor to the nucleus of brain cells — sel-based giant have synthesized a drug certain fatty-acid binding proteins— per- with an extra-strong affinity for the CB2 form the same function for THC and CBD. receptor. It was found to greatly reduce the photo by Istvan Ujvary build-up of collagen (which obstructs the ureter) in a mouse model of kidney dis- tail at the molecular level. For example, in ease. “CB2 agonists might have beneficial the late 1990s Dale Deutsch and colleagues effects in both acute and chronic kidney at Stony Brook University identified fatty disease,” the presentation by Jean-Michel acid amide hydrolase (FAAH) as the en- Adam concluded. zyme that breaks down the endogenous The trend towards studying plant com- cannabinoid anandamide within the cell. pounds and their possible therapeutic appli- In recent years Deutsch’s lab has focused cations is largely attributable to GW Phar- on the fatty acid binding proteins (FABPs) maceuticals, a British company, founded in that bring endocannabinoids from the cell 1998, that has been developing plant-based membrane to the endoplasmic reticulum medicines to treat various conditions. (where FAAH does its stuff). The ICRS scientists’ Holy Grail —even When plant cannabinoids are ingested GW Pharmaceuticals’— is a drug that ex- by people smoking or eating marijuana, erts the beneficial effects of cannabis with- THC and CBD molecules are carried in the out psychoactivity. blood into the brain, “presumably by albu- Thanks to GW providing materials and min and lipoproteins, which carry fats,” funding for studies of cannabidiol (CBD) says Deutsch. “But there’s no albumin in- ANANDAMIDE (AEA) INACTIVATION results when CBD or THC targets fatty-acid bind- and other so-called “minor cannabinoids,” side those cells in the brain.” ing proteins within the cell. At top, anandamide crosses the membrane by diffusion at the the virtual monopoly of the U.S. National In 2012 Deutsch’s lab identified the mol- cannabinoid (CB) receptor. Once inside the cell, AEA requires fatty-acid binding proteins Institute on Drug Abuse as sponsor of can- ecules that transport anandamide (and per- (FABPs) for transport through the cytoplasm to the endoplasmic reticulum (canoe-shaped nabinoid research has ended. But NIDA is haps 2-AG) in cells inside the brain: fatty- structures), where it gets broken down by fatty acid amide hydrolase (FAAH). FABP inhibi- still the biggest ICRS backer, and quite a acid binding proteins —FABPs 3, 5, and 7 tors prevent anandamide from being delivered to FAAH for breakdown, resulting in increased few presentations in Baveno were devoted to be precise. anandamide levels at the receptor. Dale Deutsch and collaborators at Stony Brook University have identified the enzyme SB-FI-26 as a potent inhibitor of the FABP transporters. to “abuse potential” and other elusive ad- To determine if these very same fatty- verse effects. acid binding proteins transport the plant NIDA funds basic research cannabinoids THC and CBD, Deutsch re- readily as anandamide and 2-AG do. why this drug exerted gender-related ef- Mostly, NIDA provides funding for sci- ported this year in Baveno, his team did Cell cultures confirmed that adding THC fects. Its pharmacology is being elucidated entists doing important basic research in three kinds of experiments. and CBD inhibited the uptake of anan- in collaboration with Roger Pertwee. physiology and pharmacology. Over the Simulations of molecular shapes, done damide and 2-AG —meaning they were years NIDA-funded scientists have figured by computational analysis, showed that binding to the same transporter molecules. Sexist Science Surpassed out the mechanisms of action by which THC and CBD “fit very nicely inside the Deutsch also cited two human studies in At the 2014 meeting, Chris Breivogel, a plant cannabinoids and endocannabinoids fatty-acid-binding-protein carriers.” which ingestion of THC or CBD was shown pharmacologist at Campbell University in (made in the body) exert their effects and Binding studies using FABPs synthe- to increase anandamide levels in the blood North Carolina, presented a paper called get metabolized (broken down). sized and purified in the lab showed that because they act as anandamide -transport “Beta-arrestin2 appears to mediate the ac- This research continues in ever-finer de- THC and CBD bind to these molecules as inhibitors. (See illustration above). tivity of cannabinoids in female mice in a Deutsch’s identification of FAAH as manner that differs from males.” the enzyme that metabolizes anandamide Beta-arrestin2 is a protein inside the cell inspired drug developers to create com- that interacts with an activated cannabi- pounds that inhibit production of FAAH, noid receptor, and —it was thought by the resulting in elevated cannabinoid levels scientists who isolated the molecule and without ingestion of exogenous (“from put arrest in its name— blocks or dampens without”) THC. Fatty-acid binding pro- the signal. teins could also be drug targets, and their Breivogel had previously shown that efficacy the topic of future ICRS talks. male beta-arrestin2 knockout mice (bred to lack the gene that encodes for beta-arres- Gender Distinctions tin2) respond to THC more strongly than Cannabinoids are more potent analgesics wild-type males. in female rats than in male rats. Rebecca Craft and MD Leitl reported in Traditionally, experiments with 2008 that the sex hormone estradiol en- hances the analgesic effects of THC in rodents have been conducted females whose ovaries had been removed, with males. whereas testosterone blocks the motion- reducing effects of THC in males. His more recent studies, using female At the 2013 meeting Aaron Haas, a post- mice, “have shown very different effects doc in Craft’s lab at Washington State Uni- from what was seen in males. The antino- versity, presented a poster showing that cieptive (but not rectal temperature) effects estradiol increases sensitivity to THC’s of THC obtained in wild-type females were anti-pain effects, but testosterone does not. nearly absent in beta-arrestin2 knockouts.” A team of Israeli researchers led by Sha- Traditionally, experiments with rodents ron Anavi-Goffer found a marked differ- continued on next page. ence in the way male and female mice respond to postnatal administration of HU-267, a cannabinoid drug developed by co-author Raphael Mechoulam. (“HU” stands for “Hebrew University.”) HU-267 is described as “a novel synthetic com- pound whose structure resembles that of ajulemic acid.” The researchers gave the Changing of the Guard among ICRS sponsors is reflected on page from the confer- drug to mice of both sexes 24 hours after ence abstract book, although the U.S. National Institute on Drug Abuse remained birth. They found that by 25 days of age, the biggest backer of the organization (and of sanctioned cannabinoid research). the males were more hyperactive while the females where more hypoactive compared A decade ago ICRS sponsors included major pharmaceutical companies. In 2014 Aaron Haas at his 2013 ICRS poster show- GW Pharmaceuticals and Otsuka (a Japanese company allied with GW) were next, with their control litter mates. ing that estradiol increases sensitivity to followed by Tilray, one of 13 Canadian companies licensed to cultivate. Now the researchers’ goal is to figure out THC but testosterone does not. —18—O’Shaughnessy’s • Winter 2015/16

ICRS from previous page

vary in the ability to cause certain effects tivation of the body’s stress system, es- (therapeutic and/or side effects). “Differ- pecially in early childhood, are known to ences in signaling, mediated by beta-arres- trigger abnormal methylation that changes tin2 and possibly other proteins, may be DNA and disables genes. So, too, in ani- why certain drugs have somewhat different mals. There have been studies that show effects in men and women,” says Breivo- differences in maternal care during the first gel. six days of a rat’s life result in different methylation patterns in promoter regions, thereby influencing gene expression. Poor diet and exposure to environmental toxins can also skew gene expression. Different responses to pain are seen in wild- 15 seconds for wild-type males, and up to 30 type and beta-arrestin2 knock-out mice on seconds, the limit of the test, for the knock- THC. In the tail-flick test, mice are treated out males. What’s in a Name? and held with the tip of the tail in a warm Knocking out beta-arrestin2 in female mice A presentation by Dr. John McPartland water bath. Untreated mice will remove their practically eliminates the effect of THC. challenged the widespread notion that tails in about two seconds, but THC will dull Wild-type females typically endured close there are three species of Cannabis —in- the uncomfortable sensation so that they to 30 seconds; the knockouts flicked out after dica, sativa, and ruderalis. leave their tails in the water longer —about about two seconds. McPartland used a novel approach in- volving “DNA barcodes.” have been conducted with males. Re- “In the knock-out males, when you re- There are 10 chromosome pairs in the searchers have known that “there are slight move the beta-arrestin2, you get an en- nucleus of every typical cannabis cell. In differences” in test results from male and hanced effect. every generation mutations occur. female animals, according to Breivogel, ‘In the knock-out females, at least in one Unlike human or plant ‘nuclear’ ge- but these were attributed to differences in kind of assay, the effect practically goes nomes, which are inherited from both an rates of metabolism by the liver, differ- away. This suggests that the beta-arrestin2 individual’s male and female parents, chlo- ences in the amount of muscle mass and/ might be involved in helping to mediate An olive orchard in Crete. roplast genomes are inherited only from or body fat, or perhaps changes in receptor the signal instead of blocking the signal.” the mother. Thus chloroplast genomes ex- level or sensitivity during the estrus cycle. Beta-arrestin knockout mice had been perience fewer mutations and evolve much It was assumed that at the receptor level, All About EVOO engineered/created and bred by Robert People in Greece, Southern Italy, and more slowly there were no differences in mechanism of Lefkowitz, a Nobel Prize-winning scientist Spain have lower rates of colon, breast, By focusing on regions of the chloro- action. at Duke University, who supplied Breivo- prostate, and ovarian cancer than Northern plast genome that are present in all plants, Breivogel explains, “Males are simpler gel with 12 animals to start breeding for his Europeans. This is attributed to differences McPartland and co-author Geoffrey Guy [to use in experiments] because research- experiments in 2003. in diet. In the Mediterranean diet, the pri- were able to calculate the degree of relat- ers don’t have to worry about changes dur- The Lefkowitz lab —working only with mary source of fat is extra-virgin olive oil edness between ‘indica’ and ‘sativa’ ge- ing the estrus cycle, and how that would males—had determined that beta-arrestin2 (EVOO); the Northerners use butter and netic sequences described in the academic affect your results... And so it just got to knockout mice had a stronger response lard. literature. be the habit where everybody just looked to morphine’s anti-pain effect than wild- at males. types. McPartland was able to create In the experiments Breivogel described Breivogel recently tested the response to Extra-virginity is important be- in Baveno, male and female beta-arrestin2 morphine of beta-arrestin2 knockout fe- cause the polyphenols in freshly baselines that expressed the genetic knockout mice, and male and female wild- males and attempted to reproduce the ef- pressed olive oil degrade with differences as a numerical value. type mice were given THC by intraperi- fects previously seen in males. “There was aging and refining. toneal injection (the most common route no difference between males and females Chloroplasts contain the genes respon- used in rodents). sible for key pieces of the photosynthetic in the effect of morphine upon deletion of The EVOO benefit is dramatic —an- al In the wild types, the males and females machinery of the plant, in addition to beta-arrestin2,” he reports, which implies most 50% lower rate of colon cancer, for showed classic symptoms of cannabinoid other genes required for the plant to sur- that the sex-differences for beta-arrestin2 example. Extra-virginity is important be- activation: a drop in body temperature, and vive. Because these genes are so crucial seen with THC may not be universal, and cause the polyphenols in freshly pressed greater tolerance for pain as measured by a for proper plant development and function, may occur only with some drugs and their olive oil degrade with aging and re-pro- tail-flick test. they mutate or “evolve” at a much slower receptors, and may even be limited to only cessing. Deleting beta-arrestin2 in the males in- rate than nuclear DNA. Comparing these a few receptor systems. That all still needs Andrea Di Francesco and colleagues in creased the effect of THC, so the knock- conserved genetic sequences between both to be investigated. Mauro Maccarrone’s lab have been study- out males showed a greater response. related and unrelated species, in addition to The differences in activation of beta-ar- ing EVOO’s mechanism of action. Expos- “What was so surprising when we did the varieties or cultivars of the same species, restin2 by THC in male and female mice ing colon cancer cells (Caco-2) to EVOO females,” Breivogel recounted, “was that it McPartland was able to create baselines are probably also present for anandamide, or an extract of its phenolic compounds re- went in the opposite direction. Instead of that expressed the genetic differences as a Breivogel says, because the differences sulted in “a selective increase in CB1 gene enhancing the effect of THC, knocking out numerical value. are also brought on by a FAAH inhibitor, expression” and “inhibited proliferation of beta-arrestin2 decreased it to practically McPartland selected genetic sequences URB597, which works by augmenting Caco-2 cells and arrested their cycle.” nothing. from Cannabis chloroplasts published in anandamide. The researchers also fed healthy rats with the academic literature, and used these “We just sort of stumbled on it one day. a standard diet and an EVOO supplement, same methods to calculate the degree of In female mice, beta-arrestin2 We were doing an experiment with a then looked for changes in cells lining the relatedness between the plants from which lab course, and I always do something I colon. Ten days of EVOO supplement led might be involved in helping to the samples were derived. The genetic dif- haven’t done yet that might be interesting. to “a significant increase in CB1 gene ex- mediate the signal instead of ferences show that the degree of variation ‘You know we’ve never looked at the fe- pression levels in colon.” between sequences was far less than those blocking the signal. male mice...’” This was due to “epigentic mechanisms.” between unrelated species. In fact, they The National Institutes of Health an- As explained by Maccarrone, “We found resembled the distance between different “The implication is that in a male when nounced a policy in May, 2014, mandating that CB1 is less expressed in cancer cells varieties as seen in other species. THC activates the cannabinoid receptor, that researchers state the sex of the animals because it is more methylated at the pro- The conclusion, therefore: Cannabis sa- beta-arrestin2 will bind to the receptor (including people) used in their experi- moter level. The gene is there, but it is not tiva and indica belong to different varieties on the inside of the cell, and interact with ments, or the sex of the animal the tissue expressed.” of the same species. The evidence is cor- other proteins to actually block the signal. or cells came from when publishing data. A “promoter” is a region of DNA that ini- roborated by the ability of all varieties of It decreases the effect of THC on the cell. Basic, pre-clinical research grant applica- tiates transcription of a particular gene. It’s cannabis —indica, sativa, and ruderalis— tions must “address the in- where DNA is turned into RNA. Methyla- to inter-mate and produce fertile offspring. fluence of sex in the design tion refers to the addition of methyl groups McPartland also called on his audience and analysis of biomedical (CH ) to a molecule. 3 to use correct terminology when referring research with animals and Catalyzed by specific enzymes, methyla- to Cannabis indica (misnamed “sativa” in cells.” tion is involved in regulating gene expres- the current vernacular), Afghanica (mis- “Biased Signaling” sion and protein function. In normal cells, named “indica”), and sativa (misnamed Drug developers hope to the promoter region is not highly meth- “ruderalis”). exploit the discovery that ylated and the gene is expressed. But in different ligands activate cancer cells, the promoter region is highly Possible Explanation of different responses to THC by male signaling pathways inside methylated and the gene is silenced. More and female mice observed by Breivogel is illustrated. In both the cell with varying ef- methylation of the gene means less CB1 males (cell at left) and females (cell at right) THC activates the ficacy. Drugs (like THC) expression and weaker endocannabinoid CB1 receptor on the outside of the cell membrane. CB1 can that activate the same re- couple to either a G-protein or a beta-arrestin at any given time. tone. ceptor in males and fe- Aberrant methylation appears to be a Both molecules couple to the same part of the receptor, so when males but then activate dif- one is there, the other is blocked. precipitating factor in the development of ferent signaling pathways Male CB1 receptors activated by THC produce an cancer. But methylation doesn’t just hap- antinociceptive (and temperature depressing) effect via inside the cell are said to pen on its own. If a gene is inappropriately G-proteins. When beta-arrestin2 is removed, THC is more exhibit “biased signaling.” methylated, then some process in the body effective. In females, Breivogel proposes, “THC may activate Such drugs might have dif- is causing this to happen. CB1 to couple beta-arrestin2 to produce antinociception, so ferent effects in males and Psychological trauma and high level ac- when you knock out beta-arrestin2, you lose the effect of THC.” females, or at least might Chloroplast structures O’Shaughnessy’s • Winter 2015/16 —19—

ICRS from previous page

Exome Sequencing two steps. First 500 genes, then all 18,000 less likely to work than a multipronged at- as a treatment for DMD would be high in It’s unusual for a single case study to genes —“and even this 18,000 genes rep- tack on the physiology of the disorder. CBDV, which —like synthetic Rimonabant represent a breakthrough, but that was resents only 1% of the genome,” McKer- G.W.’s flagship product, Sativex, a plant — is an inverse agonist at the CB1 recep- how Kevin McKernan’s presentation — nan notes. extract formulated for spraying under the tor. “Exome Sequencing of Familial Atrial Fi- As of June 2015, he adds, “For GW we tongue, has been approved by regulators in DMD is one of the so-called “orphan dis- brillation Informs Positive Treatment With are still in the 500 gene phase, and even 27 countries (starting with Canada in 2005) eases” —defined by the Food and Drug Cannabidiol”—was received by the ICRS. though there is some exciting data starting for treating pain and spasticity in Multiple Administration as affecting fewer than McKernan was the lead researcher behind to emerge, the IRBs may not approve se- Sclerosis. Sativex is in phase 3 clinical tri- 200,000 Americans— that G.W. is fo- Medicinal Genomic’s draft sequence of the quencing the whole exomes of the children als in the U.S. as a treatment for intractable cused on developing drugs to treat. Others Cannabis sativa genome in 2011. With without substantial increases to the genetic cancer pain. include Dravet Syndrome and Lennox- co-workers at Courtagen Life Sciences in counseling budget. Genes like APOE and G.W.’s Epidiolex, an almost-pure-CBD Gastaut Syndrome. Woburn, Massachusetts, he has been using BRCA1 (associated with Alzheimers and extract, is in clinical trials as a treatment A drug that is beneficial in treating the a sophisticated exome sequencing tech- breast cancer risks, respectively) are un- for two rare pediatric epilepsy syndromes most severe forms of epilepsy is likely to nique —targeting the “panel” of genes that related to pediatric epilepsy but present an (See story on page 1). be beneficial in treating most seizure dis- encode for proteins, about 1% of the whole ethical dilemma sequencing children with Brodie said that advances in high- orders. genome— to determine if certain disorders Epilepsy. Discovery of variants in these throughput screening, ever-declining se- By developing extracts and natural com- may be treatable by cannabinoids. genes can produce stress and harm to the quencing costs, and the profusion of online pounds with specified ratios, Brodie said, McKernan described a patient with an in- family and our study is designed to have databases —plus “proprietary in-house “you can form a matrix of intellectual herited form of atrial fibrillation who had no harm to the family. But even with the data”— enables G.W. to assess the role of property that will be safe... It is our belief not been helped by the conventional phar- 500 genes, we are seeing some impressive “receptors, enzymes, genes, organelles and and the belief of our commercial partners maceutical treatments —beta blockers and signals. We need more patients sequenced more” in a disease of interest. that you cannot genericize Sativex.” calcium channel blockers. By comparing to improve the signal.” One such disease is Duchenne Muscular the exome sequences of six family mem- Dystrophy (DMD), a hereditary condition Help for Acute Pancreatitis? bers, it was determined that the patient in- G.W. Pharmaceuticals’ Strategy that causes irreversible degeneration of • Acute pancreatitis is a very painful dis- herited rare mutations in calcium-channel G.W.’s drug development strategy was muscle tissue and is usually fatal before ease in which digestive cells created by genes RYR2 and CACNA1C. Knowing that outlined in a talk by James Brodie. The age 15 due to respiratory failure. acinar cells in the pancreas for use in the cannabidiol regulates intracellular calcium prevailing approach in the pharmaceutical Earlier in the conference it had been re- small intestine start digesting the pancreas homeostasis led to treatment with CBD. industry involves “screening synthesized ported by F.A. Ianotti that in a mouse mod- itself. There are no drugs to treat it —only And sure enough, sprayed in the mouth, molecular libraries to identify those most el of DMD, certain genes belonging to the painkillers. 75mg once a day, CBD promoted a regular potent and selective at a single receptor/ endocannabinoid system were upregulated Cannabinoid receptors are expressed in heartbeat. disease target.” at the time of disease onset. the pancreas and appear to prevent acinar Atrial fibrillation returned with doses be- G.W.’s approach involves figuring out Treating the mice with Rimonabant to cell pathogenesis (possibly by modulat- low 25mg given three times a day. “This the mechanism of the disease and decid- reverse the effect of CBD resulted in “a ing interacellular calcium-ion signals). case presents a private scenario,” McKer- ing which cannabis extract is best suited marked increase in locomotor activity... Using a mouse model, Huang et al tested nan aptly noted. But targeting gene panels to treating it. “Disease causation is mul- These findings indicate a novel role for GW 13542, an extract that targets CB2, to identify underlying disorders treatable tifactorial,” Brodie said, “and a ‘broad- CB1 in the development of dengerative as a treatment for acute pancreatitis and by cannabinoids is widely applicable. side’ approach may be more successful muscle disease, perhaps by affecting mus- concluded that it “eliminates intracellular McKernan’s team has been exome se- in overcoming the redundancy and multi- cle differentiation and repair processses, Ca2+ signaling in pancreatic acinar cells, quencing the DNA of pediatric epilepsy functionality that are inherent compensa- thus making this receptor a potential thera- which may provide a new therapeutic strat- patients involved in the clinical trial of tory mechanisms in biological systems. peutic target for the treatment of such dis- egy.” G.W. Pharmaceuticals’ Epidiolex. “In other words, hitting one mechanism of orders.” continued on next page The family with A-Fib was sequenced in a neurodegenerative disease or a cancer is Presumably the extract GW would deploy

From John McPartland’s ICRS Presentation Correct(ed) Vernacular Nomenclature Indica (formerly “Sativa”) Afghanica (formerly “Indica”) Sativa (formerly “Ruderalis”)

Usually feral or wild C. sativa from Europe, but Original provenance: India sometimes of Asian provenance. Central Asia (Afghanistan, Turkestan, Pakistan) Relatively short (ca. 0.6-1.5 m), densely Morphology: Relatively tall (ca. ≥1.5 m), laxly branched, Variable, depending on provenance. with narrowly lanceolate leaflets, and branched, with broad leaflets often oblanceolate, relatively sparse flowering tops. and dense flowering tops.

Physiology: Flowering time (seed germination to Flowering time short, 7-9 weeks; frost tolerance, Flowering time relatively short but variable, intiation of reproduction structures under high resin production, susceptible to mold. sometimes autoflowering; moderate frost toler- natural conditions) long, 9-14 weeks; no ance, relatively low resin production. frost tolerance, moderate resin production.

Chemistry: THC much greater than CBD; Cannabinoid profile variable (THC greater than CBD>THC; prominent terpenoids: uniquely prominent terpenoids: sabinene, or roughly equal to CBD); uniquely prominent β-caryophyllene, myrcene, imparting a flowery α-terpinolene, trans-β-ocimene, trans- terpenoids: camphene, β-myrcene, guaiol, β- fragrance. β-farnesene, imparting a flowery fragrance. and γ-eudesmol, imparting an acrid fragrance.

Psychoactivity: “Stimulating.” “Sedating.” Usually lacking.

Medical indications: Lethargic depression, nausea, appetite stimula- Insomnia, anxiety, chronic pain, joint stiffness Chronic pain, joint stiffness and inflammation, tion, migraine headaches, and chronic pain. and inflammation, muscle spasms, tremors (from epilepsy. Relative contraindications: allergy to Relative contraindications: insomnia, anxiety, multiple sclerosis and Parkinson’s disease), and cannabis. and schizophrenia. epilepsy. Relative contraindications: lethargic depression, somnolence, and schizophrenia.

Revised nomenclature was proposed by John McPartland at the 2014 meeting of are subspecies —separate varieties of one Cannabis species. McPartland traced the the International Cannabinoid Research Society. His paper, co-authored by Geof- confusion that prevails today among plant breeders and the pot-loving masses to frey Guy, used “DNA barcodes” to determine whether or not Cannabis indica and the 1970s, when a C. afghanica plant collected by botanist Richard Evans Schultes Cannabis sativa are separate species. The answer was not. C. indica and C. sativa was incorrectly identified asC. indica. —20—O’Shaughnessy’s • Winter 2015/16

ICRS from previous page

Cannabis, the anti-drug tist for Americans for Safe Access. CBD Activates Serotonin CBD Protects the RPE Ever since he started running a cannabis The U.S. contingent included Cali- The evidence now seems conclusive Macular degeneration is a leading cause dispensary in Vancouver, B.C. in 2002, fornia practioners (Jeffrey Hergenrather, that CBD works in part by activating the of blindness in the elderly. It is caused by Philippe Lucas realized that many people Michelle Sexton, William Courtney, R. 5HT1A (serotonin) receptors, as suggested accumulation in the retinal pigment epi- were using the herb as a substitute for alco- Stephen Ellis), lab directors (Jeffrey Ra- by Ethan Russo in a 2005 poster. Spanish thelium (RPE) of a compound called A2E, hol and other drugs. ber, Justin Hartzell), and political orga- researchers reported that CBD adminis- which down-regulates a compound pro- In the spirit of Tod Mikuriya’s “Mari- nizers (Steph Sherer, Martin Lee, Kristen tered in the first six hours after hypoxic- duced in the RPE called MCP-1 juana as a Substitute for Alcohol” Peskuski). There were also about a dozen ischemia had been induced in newborn Shimon Ben-Shabat and colleagues at the (O’Shaughnessy’s 2003), Lucas continued entrepreneurs seeking out scientists with piglets had strong neuroprotective effects. University of the Negev have determined collecting data from patients who used products to market and/or expertise to tap. But if given along with a compound that that cannabinoids (HU-210, HU-308 and cannabis as an alternative to harder drugs. blocks 5HT1A, there is no beneficial -ef CBD) counter the down-regulation of He has been updating his findings at ICRS “With popularity of a name,” fect. MCP-1 and provide neuroprotection. meetings since 2005, in ever more impres- Raber noted, “comes the greater sive posters. three discoverers In Baveno he gave a talk based on a sur- potential for its abuse by those vey to which 628 Canadians had respond- simply seeking to capitalize on ed. Consisting of 414 questions [that’s a lot of questions], it was distributed online and a transaction involving the cul- in hardcopy to patients. Lucas found: tivar.” “Overall, 86.6% of patients reported sub- stituting cannabis for at least one other Raber gave an oral presentation, showing substance: 80.3% (n=504) of patients stat- with data from his lab, the Werc Shop, the ed that they used cannabis as a substitute extent to which cannabis sold in Califor- for prescription drugs, 51.7% used it as a nia and Washington dispensaries is inaccu- substitute for alcohol, and 32.6% used it as rately named. “With popularity of a name,” a substitute for illicit substances. Raber noted, “comes the greater potential “The main reasons cited included ‘better for its abuse by those simply seeking to symptom management’ and ‘less adverse capitalize on a transaction involving the side effects.’ Patients who listed a greater cultivar.” number of symptoms were more likely to Raber also found lamentable inconsis- report cannabis substitution, and younger tency in the way cultivars were dubbed patients (below 30) were far more likely to indica and sativa. “A specifically named substitute cannabis for prescription drugs, cultivar at one disepnsary is not necessarily the same product in the package at another alcohol and illicit substances than older pa- in order of their landmark findings: William Anthony Devane (right), molecular pharma- tients (50 and over).” dispensary simply because it posesses the cologist. Working in the lab of Allyn Howlett at St. Louis University in 1988, he discovered Lucas ended with a call for “research into same name. This may also be the case even the cannabinoid receptor (dubbed the CB1 receptor when a second one was found in 1992). cannabis as a treatment for problematic week to week at the same dispensary, lead- Lumír Ondřej Hanuš (left) analytical chemist. Working with Devane in the lab of Raphael substance use in non-patient populations.” ing towards many different reports of the Mechoulam at Hebrew University in Jerusalem, he isolated the first endocannabinoid on physiological impacts for a specific name March 24, 1992. Devane named it “anandamide,” incorporating the Sanskrit word for “bliss.” Bringing it all back home and considerable numbers of frustrated pa- Shimon Ben-Shabat (center), medicinal chemist. Working in Mechoulam’s lab in 1994, he The 80th and final oral presentation in tients seeking to find relief with a specific was the first to isolate the endocannabinoid 2-arachidonoyl glycerol (2-AG). Baveno was by Jahan Marcu, PhD, who varietal.” This photo was taken at the 2014 ICRS meeting with Dr. Ben-Shabat’s cellphone by a pass- erby. Hanuš describes it as “a unique picture,” the first of the three discoverers together. “It has gone from lab research to auditing can- Raber foresees more accurate identifica- nabis production and distribution by U.S. is for us pleasant to see how these three discoveries influenced science,” he says. “This ICRS tion of cultivars based on terpene content. conference is basically on cannabinoid receptors and endocannabinoids. Around the world growers and dispensaries as Senior Scien- there are whole laboratories on this subject.” Will the industry accept? ASA’s Audacious Audit Offer As the cannabis industry burgeoned in The AHP set to work on a monograph es- Marijuana prohibition can be seen the ICRS meeting and gave us the back recent years, naturopath Michelle Sexton tablishing standards of purity for cannabis as part of a broader war on botani- story on the audit project. Researching the urged ASA’s executive director, Steph that could be incorporated into regulations origins of marijuana prohibition, she came Sherer, to push for safety standards that by state legislators. (AHP standards are cal medicine. to see it in the context of “scientific medi- would protect patients’ interests. Sexton widely used by companies that make and cine” wiping out alternative approaches suggested involving the American Herbal distribute licensed herbal supplements.) is another area that often calls for improve- —including herbal medicine— in the first Products Association —AHPA, a trade as- Sherer raised funds for the mongraph’s ment. “The rules and requirements dif- part of the 20th century. sociation for the natural-products indus- publication in December, 2013, a revised fer from state to state,” Marcu says, “but In 1910 the Carnegie Foundation funded try— and the American Herbal Pharmaco- edition issued in the fall of 2014, and a you don’t throw away moldy cannabis or a crtique of U.S. medical schools by a lay- peia —AHP, which publishes monographs second volume —a “Therpaeutic Com- outdated products into an easily accessible man named Abraham Flexner (who also defining “standards of identity, purity, and pendium” citing all the medical literature, trash can.” Marcu says AHP standards are had connections to the Rockefeller family analysis for botanicals.” coming soon. not costly to comply with. and Johns Hopkins University). It was a Sherer pursued them. AHPA agreed to blueprint for transforming the profession. Auditing the producers Facilities that pass the audit get a label create guidelines on proper manufacturing, The American Medical Association be- At the 2014 ICRS meeting, ASA’s Jahan vouching for the quality of their operation, processing, dispensing, and lab procedures. came the mechanism for driving out com- Marcu described how an “audit” is con- be it cultivation, manufacturing, dispens- petition from herbalists, homeopaths, and ducted to confirm that a facility —a farm, ing, or lab reports. “Doctors can’t tell peo- all other practitioners who had not been dispensary, or lab— meets AHP standards, ple where to get cannabis,” Marcu notes, trained at elite medical schools (whose labs complies with state and local regulations, “but they can remind patients to look for and hospitals required underwriting by the and qualifies for “Patient-Focused Certifi- the PFC seal. If you have this seal on your wealthy, and whose tuition fees effectively cation.” product, you know that it’s following basic excluded working-class students). “I go to the site,” says Marcu. “I make safety and handling protocols for botanical ‘Scientific medicine’ glorified technol- sure that the place is clean. I go through medicine, or that the readings from the lab ogy and research. Historian Richard Brown all their paperwork —staff training manu- are going to be within acceptable limits of accuracy.” attributes its credibility to the work of Eu- als, documentation. Is there a logbook? Do ropean bacteriologists who had “identified they have policies and procedures stating Certification by a third party is common in U.S. industries. “FDA does not certify discrete, external, and specific agents of how they do things? When was the last disease. This perspective encouraged the time they applied a pest-management prod- manufacturing facilities and labs,” Marcu points out. “They punt it to third parties idea of specific therapies to cure specific uct? How much do they apply? What batch pathological conditions, and it diverted number of what pesticide product did you that are often established by the very in- dustry they’re trying to regulate.” Marcu attention from the social and economic use? When was the last test on the water causes of disease.” you’re using from your well? When was compares his role to that of a rabbi certify- ing a food product as kosher. Scientific medicine made possible the your last soil test? Where did the last batch cannabis prohibition of 1937 (and its con- you cultivated go and could you initiate a Marcu’s presentation to the ICRS in July 2014 framed the ASA audit as an experi- tinuation to this day) by disrespecting recall? Is the facility processing on site? “crude” herbs. Scientific medicine recog- AHP Monograph cover features a photo of ment, and as this issue goes to press in De- Are solvents stored properly and labeled? nizes that certain plants contain specific the garden at the University of Mississippi Are fertilizers and fuels stored properly cember, 2015, the results are inconclusive. where Cannabis is grown legally under fed- “Everyone really seems to like the audit active ingredients that can be isolated, syn- eral law (surrounded by a 10-foot tall barbed and labeled?” thesized, and marketed as medicine. Marcu typically sees problems that can and certification except for businesses,” wire fence, with cameras and a guard tower he said. “The patients like it, regulators, Marijuana prohibition can be seen as part for extra security). The herb is for distribu- be readily resolved. Most common at the researchers love it... We’ll just have to see of a broader war on botanical medicine. tion by NIDA to government-sanctioned re- dispensary level, he says, is “the lack of a searchers. The grower, Mahmoud ElSohly, plan to record and report adverse events.” about the industry.” is one of the Monograph editors. Waste disposal by labs and dispensaries Steph Sherer had come to Baveno for