FROM:

Matthew Schnur, UCCS Student Cell & Molecular Biology

________________

Dr Robert Melamede, UCCS Chair (retired)

________________

Warren Edson, Esquire

________________

BY HAND

Date Delivered:

Dennis E. Ellis, Executive Director

Colorado Department of Health and Environment

4300 Cherry Creek Drive South

Denver, Colorado 80246-1530

Re: Petition to Add Types 1 and 2 Diabetes Mellitus to List of Debilitating Medical Conditions Pursuant to Colorado Constitution, Article XVIII § 14 and 6 CCR 1006-2

On behalf of the undersigned physicians and patients, we hereby submit the enclosed petition, pursuant to 6 CCR 1006-2, to add both Type 1 and 2 diabetes to the list of debilitating medical conditions for which the medical use of marijuana is authorized under the Colorado Constitution, Article XVIII § 14. 6 CCR 1006-2, Regulation 6, section D states:

“Beginning June 1, 2001, the department shall accept physician or patient petitions to add debilitating medical conditions to the list provided in paragraphs A and B of this regulation. The department shall determine if a public rulemaking hearing to modify this regulation is appropriate, and if so, shall petition the Board of Health to set a date for such hearing within one hundred twenty days of receipt of the patient or physician petition. If the department determines that a public rulemaking hearing is not appropriate, it shall notify the petitioner of its action within one hundred eighty days of receipt of submission of the petition. In making its determination, the department will consider whether there is information that the proposed condition is chronic, debilitating, and may be specifically diagnosed, and whether there is scientific evidence that treatment with marijuana may have a beneficial effect.”

I. Introduction:

In the following discussion we intend to prove that diabetes mellitus is a clearly diagnosable disease with specific, easily utilized tests that demonstrate exact parameters for categorization into one of two subtypes. These diagnostic criteria have been developed by the world’s leading experts in diabetes research; the World Health Organization (WHO) and American Diabetes Association (ADA). Second, we shall identify symptoms and complications resulting from the chronic progression of this disease. In this section we will also address the evidence demonstrating Types 1 and 2 diabetes mellitus as chronic. Three of the symptoms known to occur in the diabetic state, have already been accepted in 6 CCR 1006-2, as acceptable criteria to recommend the use of medical marijuana for (cachexia, severe nausea, severe pain). On these grounds alone, pursuant to the definition of debilitating medical condition in Regulation 5, section B, the use of medical marijuana for diabetes should be recognized. In addition to alleviation of debilitating symptoms of diabetes mellitus outlined in 6 CCR 1006, the medical use of marijuana can prevent nerve damage, blindness, amputation, ketoacidosis, and insulin resistance; all of which are conditions that further reinforce the concept that diabetes is debilitating. The rest of our discussion shall focus on the scientific research identifying specific molecular mechanisms of therapeutic benefit from intervention of diabetes mellitus with medical marijuana.

As marijuana is a whole plant medicine, each dosage will vary with active ingredients, and thus, will never be allowed in clinical FDA trial. In light of this fact, it would be unreasonable to insist upon clinical trial data for support of this petition. Indeed, if clinical trial data was available, petitions such as these would not need to be written. With this in mind, we intend to demonstrate that medical marijuana “MAY HAVE A BENEFICIAL EFFECT” for diabetes by utilizing rodent, human, in vivo, and in vitro studies. While none of these models taken individually can account for substantial therapeutic validity, the culmination of consistent findings between species, data analysis, in vitro cultures, and whole organism studies, characterizes a complex system of hormonal interaction by which cannabinoids benefit diabetes patients at a multitude of levels. Additional benefits in prevention and treatment of microangiopathies, sexual dysfunction, hypertension, inflammation, poor wound healing, ketoacidosis, advanced glycation end products, and gastroparesis will also be addressed. Taken as a whole, this presentation overwhelmingly demonstrates beyond reasonable doubt that Diabetes mellitus meets all necessary criteria for its inclusion into diseases for which medical marijuana be allowed state approval.

II. Diagnosis of Diabetes Mellitus:

According to the WHO, diabetes mellitus is “a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion, insulin action, or both”[1]. Initial presentation is associated with polyuria, thirst, blurred vision, and weight loss. Criteria for diagnosis have not changed since 1985[2], and involve glucose concentration cut-off values for various biological matrices according to the following guidelines when testing 2h postprandial to a standardized glucose load:

Whole Blood: venous >180mg/dL capillary > 200mg/dL

Type 1 and Type 2 diabetes mellitus are further subsets of classification. Type 1 diabetes mellitus now encompasses the terms juvenile onset and insulin dependent (IDDM), whereas Type 2 now refers to NIDDM and adult onset. Type 1 diabetes mellitus results from complete β-cell destruction and is considered an autoimmune process. Autoantibodies against glutamic acid decarboxylase, a β-cell specific enzyme, are found to exist in up to 95% of Type 1 patients. Type 1 diabetics also have no detectable insulin or C-peptide levels, whereas Type 2 diabetics will still have detectable insulin plasma levels[3]. Type 2 diabetes mellitus is characterized by impaired insulin secretion or impaired insulin signaling[4] [5]. A strong correlation exists between obesity and Type 2 diabetes[6]. More specifically, Type 2 diabetes is correlated to fat tissue density, as Type 2 patients not diagnosed as obese will predominantly still feature excessive fat distribution in the abdominal cavity[7]. Both forms of diabetes mellitus are associated with genetic predispositions[8] [9], and thus when clinical presentation occurs, can be considered a chronic condition. Despite having different etiologies, the WHO recognizes homologous clinical stages of progression of both types of diabetes mellitus. Furthermore, as the next sections statistics demonstrate, the ADA validates extrapolation of epidemiological data from Type1 complications to those of Type 2.

III. Diabetes is Debilitating and Chronic:

The following statistics were taken from the American Diabetes Association (http://www.diabetes.org/diabetes-statistics/complications.jsp), unless otherwise cited, and prove unequivocally that diabetes is a debilitating and chronic condition.

1. Heart disease and stroke

• Heart disease and stroke account for about 65% of deaths in people with diabetes.
• Adults with diabetes have heart disease death rates about 2 to 4 times higher than adults without diabetes.
• The risk for stroke is 2 to 4 times higher and the risk of death from stroke is 2.8 times higher among people with diabetes.
• About 73% of adults with diabetes have blood pressure greater than or equal to 130/80 millimeters of mercury (mm Hg) or use prescription medications for hypertension.

2. Blindness

• Diabetic retinopathy causes 12,000 to 24,000 new cases of blindness each year making diabetes the leading cause of new cases of blindness in adults 20-74 years of age.
• ¾ of all diabetic patients 15+ years have retinopathy[10].
• Nearly all individuals diagnosed before the age of 30 will develop retinopathy within the first 20 years of having the disease[11].

3. Kidney disease

• Diabetes is the leading cause of kidney failure, accounting for 44% of new cases in 2002.
• In 2002, 44,400 people with diabetes began treatment for end-stage renal disease (ESRD).
• In 2002, a total of 153,730 people with ESRD due to diabetes were living on chronic dialysis or with a kidney transplant.
• In people with type 1 diabetes, therapy that keeps blood glucose levels as close to normal as possible reduces damage to the kidneys by 35% to 56% (New England Journal of Medicine, September 30, 1993). Experts believe that these results can also be applied to those with type 2 diabetes.

4. Nervous system disease

• About 60% to 70% of people with diabetes have mild to severe forms of nervous system damage. The results of such damage include impaired sensation or pain in the feet or hands, slowed digestion of food in the stomach, carpal tunnel syndrome, and other nerve problems.
• Almost 30% of people with diabetes aged 40 years or older have impaired sensation in the feet (i.e., at least one area that lacks feeling).
• Severe forms of diabetic nerve disease are a major contributing cause of lower-extremity amputations.
• More than 60% of nontraumatic lower-limb amputations occur in people with diabetes.
• In 2002, about 82,000 nontraumatic lower-limb amputations were performed in people with diabetes.
• The rate of amputation for people with diabetes is 10 times higher than for people without diabetes.
• Diabetic polyneuropathy was found to occur at a rate of 30% within the diabetic population[12]

5. Sexual Dysfunction

• Men with diabetes are 2 times as likely to experience erectile dysfunction as men without diabetes.
• Women with type 1 diabetes are twice as likely to experience prevalence of sexual dysfunction compared with women without diabetes.

6. Other complications

• Uncontrolled diabetes often leads to biochemical imbalances that can cause acute life-threatening events, such as diabetic ketoacidosis and hyperosmolar (nonketotic) coma.
• People with diabetes are more susceptible to many other illnesses and, once they acquire these illnesses, often have worse prognoses. For example, they are more likely to die with pneumonia or influenza than people who do not have diabetes.
• 76% patients surveyed have severe chronic abdominal discomfort, with 29% reporting nausea & vomiting, and 34% having abdominal pain[13].
• Gastroparesis occurs in roughly 50% of all diabetics[14]

7. Evidence for Cachexia in Diabetes Patients

The following excerpt was taken from a clinical study[15] on the only known case of reversibility in nerve conduction damage in diabetes, but also illustrates the severity of the diabetic condition.

“A 36-year-old woman presented with subacute hyperglycemic symptoms. Soon after initiation of insulin therapy and the decline of HbA1c from 14.9 to 5.5%, she developed severe lancinating pain and profound weight loss associated with anorexia, amenorrhea, insomnia, and dehydration. On examination, allodynia was so pronounced that a light touch to her shoulder would cause her to weep. Profound loss of subcutaneous adipose tissue and loss of muscle bulk was evident, such that her weight had decreased from a baseline of 58.3 to 41.8 kg (corresponding to a decrease in BMI from 21 to 15.7 kg/m2). Pain, temperature, and light touch sensation were abnormal in the hands and feet.”

8. Preventing diabetes complications

• Studies in the United States and abroad have found that improved glycemic control benefits people with either type 1 or type 2 diabetes. In general, every percentage point drop in A1C blood test results (e.g., from 8.0% to 7.0%) reduces the risk of microvascular complications (eye, kidney, and nerve diseases) by 40%.
• Blood pressure control reduces the risk of cardiovascular disease (heart disease or stroke) among persons with diabetes by 33% to 50%, and the risk of microvascular complications (eye, kidney, and nerve diseases) by approximately 33%.
• In general, for every 10 mm Hg reduction in systolic blood pressure, the risk for any complication related to diabetes is reduced by 12%.
• Improved control of cholesterol or blood lipids (for example, HDL, LDL, and triglycerides) can reduce cardiovascular complications by 20% to 50%.

As kidney disease, heart disease, blindness, retinopathy, glaucoma, amputation, abdominal pain, nerve damage, erectile dysfunction, hypertension, loss of sensation or heightened pain sensation, and gastroparesis are common complications of diabetes, we find ample documentation to satisfy the requirement that diabetes be debilitating. Furthermore, the documentation citing diabetes as a disease characterized by chronic vomiting and nausea, as well as severe abdominal pain, and in some circumstances cachexia, demonstrates it as a disease that already satisfies three symptoms that medical marijuana use is legally permitted for. In addition, our discussions will demonstrate that marijuana can lower blood pressure, improve sexual physiology, prevent microangiopathies, and restore normal insulin signaling.

IV. Scientific Evidence Supporting Use of Medical Marijuana for Diabetes Mellitus

Control of glucose levels has unanimously been demonstrated as the best means to prevent secondary complications of diabetes mellitus. Thus, pharmaceutical agents that can sensitize the body to insulin signaling, normalize insulin secretion, reduce agents that inhibit insulin signaling, or inhibit toxic by-products of hyperglycemia, are the most beneficial treatments for the diabetic state. As we shall demonstrate, the use of marijuana has therapeutic benefits at every level previously mentioned. Before this discussion, a basic review of insulin signaling is required.

Upon binding to the heterotetrameric insulin receptor (IR), insulin causes tyrosine autophosphorylation of the receptor’s β-subunits[16], which consequently results in dissociation of the activated insulin receptor substrate-1 (IRS-1)[17]. IRS-1 also retains sites for tyrosine phosphorylation that become occupied during IR autophosphorylation[18]. Once dissociated from the IR receptor, IRS-1 transfers its P_i to PI3K [19], a process that is responsible for the translocation of GLUT-4 to the plasma membrane. The IRS-2 is similarly responsible for the activation of PI3K, however, only activates after prolonged IR tyrosine autophosphorylation and requires a higher cytosolic concentration[20]. In states of excess insulin activity at the IR, autophosphorylation of both serine and threonine sites can occur as a means to down regulate activation of PI3K via IRS-1 or IRS-2[21] [22]. In addition to inhibitory phosphorylation sites as a down-regulatory mechanism to PI3K activation, both IRS-3 and IRS-4 will inhibit PI3K-mediated GLUT-4 translocation[23]. In a separate cascade, autophosphorylation of the IR is responsible for the Ras/Raf initiated activation of ERK1/2, also know as MAPKp42/p44. Ultimately, this sequence is an amplification mechanism to activate various transcription factors and nuclear receptors. Most notable of these nuclear proteins is the PPARγ receptor, which is responsible for a multitude of IR mediated glucose and lipid metabolizing effects. The most utilized class of pharmaceuticals in the treatment of Type II diabetes, thiazolidinediones (TZDs) exert their insulin sensitizing influence by binding with high affinity to the PPARγ. The PPARγ is a class II nuclear receptor, meaning it will dimerize with the retinoic X receptor. Activation of both PPARγ and retinoic X receptors has been demonstrated to lower glucose plasma levels[24] [25]. In addition, PPARγ activity can inhibit gene transcription of TNFα, plasminogen activator-inhibitor-1, leptin, resistin, and interleukins -6 and -11, all of which have been found to increase insulin resistance[26]. Furthermore, transcription of insulin-IR sensitivity enhancing proteins adiponectin, fatty acid transport protein, and IRS-2, are all up-regulated by PPARγ activation.

1. THC Reduces Progression of Diabetes in Rodent Models:

The streptotozocin (STZ) virus is administered to rodents being utilized in studies investigating pharmacological actions of antidiabetic agents in models of Type 1 DM. When given at higher doses (200mg/kg), STZ employs a rapid cytotoxic response against β-cells, which uniquely express the glutamic acid decarboxylase enzyme[27]. Employing gradual doses of STZ (5-40mg), researchers designed a mouse model of Type 1 DM that parallels that of humans in that hyperglycemia is slow in onset, with gradual progression of destruction to the pancreas resulting from lymphocytic infiltration[28]. Mammalian immunological studies on Type 1 DM find an imbalance in the TH1/TH2 profile, with up-regulation of TH1 and its characteristic cytokines[29] [30]. When THC was given gradually to STZ infected mice, mRNA levels of the TH1 cytokines TNFα, IL-12, and IFN-γ were significantly decreased²⁷. THC was also responsible for slowing the progression of elevated serum glucose and inhibiting the loss of insulin secretion, as compared to controls. As we shall see in the next section, the inflammatory response in conjunction with hyperlipidemia, are the primary causal factors in hyperglycemia, resulting in nearly all secondary complications of the diabetic state.

2. Lipids, Inflammatory Cytokines, and Diabetes:

While there are differences in insulin resistance mechanisms between muscle and adipose tissue, it is apparent the underlying pathology causing improper IR signaling is due to both an inflammatory and hyperlipidemic state. Increased concentrations of both FFAs in muscle tissue and their respective metabolites, long chain acyl-CoA, diacylglcerol (DAG), and triglycerides, have been correlated with decreased insulin signaling in the rat[31]. Insulin resistance due to elevated plasma FFA levels follows homologous mechanisms between humans and other mammals[32] [33] [34] [35] [36]. More specifically, prolonged lipid exposure initiates insulin resistance in both fast- and slow-twitch muscle fibers studied in vivo from both human and rat studies[37] [38] [39]. Increased FFAs inhibit insulin mediated glucose disposal mechanisms of both oxidative and non-oxidative origins[40]. In both humans and rats, infusion of FFAs or high fat diets are associated with the insulin resistant state[41] [42] [43].

As previously mentioned, the IR cascade involves tyrosine autophosphorylation. DAG, TAG, and other by-products of FFA metabolism have been shown to activate the intercellular stress kinases JNK and PKCθ, both of which activate serine residues located on the IRS-1[44] [45] [46] [47] [48] [49]. A study was conducted on human 3T3-L1 adipocytes utilizing various mixtures of both saturated & unsaturated FFAs at physiologic concentrations[50]. Lysates were compared to controls via several immunoblotting techniques and SDS-PAGE. In this study, FFAs inhibited IRβ tyrosine autophosphorylation and caused a 40% decrease in IRβ expression levels. Near homologous data was found for FFA activities against IRS-1. Similar inhibitory effects on AKT/PKB were found, however, no changes in total protein levels were observed. Other studies also identify decreased PKB phosphorylation in rat soleus muscle[51], as well as inhibited IRS-1 & -2 mediated PI3K and AKT activation during in vivo FA infusion studies in mammals[52] [53]. An assay has been developed to identify inhibitors of GLUT-4 translocation[54]. Treatment of 3T3-L1 adipocytes with FFAs at 500μM for 3 hours resulted in complete inhibition of GLUT-4 translocation. Furthermore, FFA treatment at 1mM for 1 hour, .5mM for 3 hours, and .3mM for 6 hours, results in a 70-90% inhibition of IR mediated glucose uptake initiated by 1.7mM of insulin.

While FFA accumulation is found to cause insulin resistance via activation of PKCθ and its consequent serine phosphorylation of IRS-1 & -2, the most profound effects on insulin mediated glucose metabolism are seen from inflammatory cytokines. A high correlation between elevated FFAs and TNFα have been found in the diabetic state[55]. TNFα of adipose origin is increased in both humans and rodents in obesity related insulin resistant states[56] [57]. Increased mRNA expression levels of TNFα within adipose tissue are directly correlated to hyperinsulinemia[58]. This inflammatory cytokine is elevated to 2.5x normal concentration in obesity[59], and is directly linked to a multitude of pathological mechanisms underlying insulin resistance[60]. The link between insulin resistance, elevated FFAs, and increased inflammatory cytokines is an up-regulating process. Both TNFα and FFAs increase JNK expression[61]. JNK itself can cause insulin desensitization, as it initiates direct phosphorylation of ser307 of IRS-1[62]. In both diet-induced and genetic rodent models of obesity, pharmacologically induced JNK deficiency enhanced insulin signaling and sensitivity[63]. Hepatic JNK suppression inhibits regulatory enzymes of gluconeogenesis[64]. JNK and IKKβ phosphorylation are exponentially increased during FFA treatments⁴⁹. Upregulation of TNFα gene expression is the result of increased JNK phosphorylation[65]. ELISA analysis revealed that TNFα secretion increases 80% with FFA treatment in 3T3-L1 adipocytes⁴⁹. The same group also found restored glucose metabolism in JNK adipocyte knockouts treated with the same levels of FFAs. TNFα in turn, has been demonstrated to elevate plasma FFAs[66] [67]. This phenomenon is in part due to lipolysis. TNFα decreases perilipin levels[68]. Perilipin inhibits hormone sensitive lipase (HSL) adhesion to fat droplet surfaces, where HSL is responsible for lipid metabolism. TNFα induced lipolysis is known to occur in rat, mouse, and human adipocytes[69] [70] [71] [72]. Thus, we see a cyclic upregulation of the hyperlipidemic-pro-inflammatory cytokine state in insulin resistance.

TNFα is by far the most inhibiting factor of insulin mediated glucose metabolism both in vivo and in vitro[73] [74], in animal models[75] [76] [77], and in humans[78]. At a macroscopic level, TNFα inhibits both peripheral glucose uptake as well as insulin mediated suppression of hepatic gluconeogenesis[79]. TNFα also regulates several important cytokines in insulin resistance. Of most importance is the TNFα mediated inhibition of adiponectin secretion[80] and gene expression levels in both immature and fully differentiated 3T3-L1 adipocytes[81] . Adiponectin is a skeletal muscle tissue insulin sensitizing adipokine found in both humans and rodents. TNFα also causes increases in insulin desensitizing cytokines. Suppressor of cytokine signaling -1 & -3 (SOCS-1/-3) are known to mediate several aspects of TNFα induced insulin resistance[82] [83] [84]. In white adipose tissue, SOCS-3 levels are increased over a prolonged duration as a result of elevated TNFα⁸². SOCS-1 & -3 are known to cause IRS-1 &-2 breakdown via the ubiquitin pathway[85]. Both tyrosine phosphorylation of IRS-1 and its ability to activate PI3K are inhibited by elevated levels of SOCS-3 in a COS-7 cell line⁸². SOCS-1 &-3 are both responsible for elevated FA synthesis via activation of the SREBP-1c transcription factor[86].

TNFα is also responsible for direct actions on IR signaling. It has been shown to decrease both IRS and GLUT-4 protein levels[87]. Inhibition of autophosphorylation within the IR is seen in studies employing both “low dose chronic”[88] and “short term incubation”[89] of adipocytes with TNFα. This inflammatory cytokine is known to inhibit tyrosine phosphorylation sites of the IR, all the IRS proteins, and protein phosphatase-1[90] [91]. In human adipocytes, rat hepatocytes, human fibroblast NIH-3T3 cells, and embryonic human 293 kidney cells, TNFα is shown to decrease tyrosine phosphorylation of both IR and IRS-1[92] [93] [94]. Whether by serine phosphatase inhibition, serine kinase activation, or a combination of both, it has been demonstrated that TNFα induces serine phosphorylation of IRS-1 with devastating results to glucose metabolism[95]. AN example of this is the IRS-1 conversion into an IR tyrosine kinase inhibitor[96]. In addition, these serine activated IRS-1 proteins are extremely unstable and quickly degrade[97].

TNFα causes changes in adipocyte differentiation, gene expression, and protein levels that ultimately result in improper lipid and glucose metabolism. The fully differentiated 3T3-L1 human adipocyte expresses both Glut-1 and -4, however, only Glut-1 is transcribed in preadipocytes[98] [99]. TNFα prevents preadipocyte development in both 3T3-L1 cells and other preadipocytes[100] [101] [102]. TNFα treatment (.04nmol/I/24h) reduced ACRP30 gene expression by 27%[103]. ACRP30 is a well documented adipokine that inhibits hepatic gluconeogenesis, enhances skeletal muscle FA oxidation, and interestingly, enhances weight loss without an anorexigenic response[104] [105]. The same study utilized oligonucleotide microarray analysis of 3T3-L1 human adipocytes following TNFα treatment. Downregulation occurred at rates of -3.4x for CEBPα, -2.3x for RXRα, and -2.0x for PPARγ. CEBPα restricts growth arrest in mature adipocytes and facilitates metabolism[106] where it is highly expressed and its isoform, CEBPβ is suppressed[107]. CEBPβ expression was increased 1.6x during TNFα treatment. Dimerization of CEBPβ and NFκβ, a well known central mediator to a multitude of inflammatory pathways, is known to occur and increase gene expression(Hotam, 1995). Other significant protein expression alterations included an 8-fold increase in iIKK, the rate limiting protein for Iκβ degradation[108]. Taken in combination with the discovery that TNFα facilitates NFκβ translocation from the cytoplasm to the nucleus, we see yet another mechanism by which TNFα induces a broader-range inflammatory response.

TNFα also inhibits metabolism and insulin signaling in skeletal muscle. Skeletal muscle constitutes the major target tissue for Type 2 DM insulin resistance[109]. FA and glucose metabolism in skeletal muscle is tightly regulated by AMP kinase[110]. AMPK is an enzyme that phosphorylates acetylCoA carboxylase (ACC), which enhances FA oxidation in skeletal muscle[111] [112]. AMPK activity has also been correlated with increased mitochondrial biogenesis, an important factor in FA oxidation[113]. Activating AMPK with the agonist AICAR has been demonstrated to facilitate glucose metabolism in a Wortmannin (PI3K antagonist) inhibiting fashion[114] [115] [116]. AMPK activity is allosterically enhanced with an increase in the AMP: ATP levels[117] [118] [119], while protein phosphatase-2 (PP2-C) is known to mediate AMPK dephosphorylation[120]. Exercise has also been demonstrated to enhance AMPK activity[121] [122] [123]. This effect is attributed to the insulin sensitizing effects of exercise observed in animals and humans with Type 2 DM[124] [125]. TNFα causes both de-activation of AMPK via a 27% reduction in ACC phosphorylation, as well as causing an up-regulation of PP2-C and decreased Thr172 phosphorylation within the active site of AMPK[126].

Besides TNFα underlying a multitude of insulin desensitizing mechanisms, other inflammatory adipokines/cytokines have been found to cause additional metabolic deficiencies in the diabetic state. Interleukin-6 (IL-6) has been found to cause insulin resistance through several actions in humans[127]. IL-6 is a proinflammatory adipokine found elevated in states of glucose intolerance, obesity, and Type 2 diabetes[128] [129] [130] [131] [132] [133]. Increased levels of IL-6 have been correlated with risk of developing Type 2 DM[134]. Utilizing RT-PCR and various immunoprecipitating techniques, it has been demonstrated that IL-6 reduces expression of IRS-1 and GLUT-4 by 35%, as well as decreased both GLUT-4 and PPARγ mRNA levels[135]. The same group also identified an up-regulation mechanism of IL-6 by TNFα, both of which regulate the JAK-STAT pathway. Both IL-6 and TNFα inhibit IRS-1 tyrosine phosphorylation via preferential ser307 phosphorylation mediated by the activation of SOCS-3[136] [137]. Furthermore, a second group identified an inhibitory effect against Akt and PI3K activation, in addition to IRS-1 inactivation[138]. In both rodent and human 3T3-L1 adipocytes, IL-6 inhibits lipoprotein lipase (LPL) activation[139]. Enhanced activation of LPL has been demonstrated to relieve insulin resistance[140] [141]. This enzyme regulates the rate limiting step of hydrolyzing lipoproteins abundant in triglycerides[142].

3. Cannabinoids Enhance FFA & Glucose Metabolism by Reducing the Pro-Inflammatory State.

Cannabinoids can enhance FFA and glucose metabolism via activation of PPARγ[143] [144] [145]. Reversal of the diabetic state has been correlated to PPARγ activation[146]. Like cannabinoids, oxyiminoacetic acid derivatives and the previously mentioned TZDs are pharmaceutical agents that exert an antidiabetic activity via activation of PPARγ[147] [148] [149] [150]. The most widely distributed antidiabetic drugs in the world are TZDs[151] [152]. Common side effects of TZDs include both weight gain and toxicity¹⁴⁸. This weight gain may in fact reduce FFAs by incorporation into adipocytes undergoing differentiation. PPARγ facilitates early differentiation of fibroblasts into preadipocytes[153], adipogenesis[154], and late stage maturation[155]. As a transcription factor, PPARγ enhances FFA metabolism by upregulating FABP, LPL, acyl-CoA synthase, adiponectin, fatty acid transport protein (FATP), and IRS-2[156] [157]. In addition, PPARγ inhibits transcription of the insulin desensitizing factors TNFα, PAI-1, resistan, leptin, and IL-6 & -11²⁶.

Within the brain, cannabinoids can also increase metabolism of glucose and FFAs via activation of the AMPK enzyme[158] that, as previously discussed, is inhibited by inflammatory cytokines and FFAs. This in turn may prevent hyperglycemic pathology in the CNS. Cannabinoid inhibition of inflammatory mediators may also enhance the activity of AMPK in peripheral tissues, although no studies on the diabetic state and treatment with cannabis have focused on AMPK activation.

Cannabinoids also impart a potent anti-inflammatory response characterized by inhibition of various cytokine pathways. We previously discussed the shift in the Th1/Th2 ratio, with an increase in Th1 cells and their respective inflammatory cytokines in the diabetic state. Cannabinoid treatment under variable experimental conditions has been found to bring the Th1/Th2 profile into equilibrium, and in other circumstances, to increase Th2 and decrease Th1[159] [160] [161] [162]. Inflammatory cytokines involved in insulin resistance of multiple origins have unanimously been demonstrated to be down regulated by THC. THC decreases TNFα production or expression from human and mouse macrophage cell lineages[163], human in vitro NK cells[164] [165], and with CBD in peripheral blood mononuclear cells[166]. Cannabinoids have also been shown to inhibit IL-1, -6, -10, and -12^{162 [167]}.

4. Cannabis, Inflammation, VEGF, and Retinopathy

In addition to inflammatory cytokines being elevated in the diabetic state, COX-1 and -2 synthesized inflammatory mediators are produced in excess from arachidonic acid[168] [169]. Indeed, several thromboxanes, prostacyclins, and prostaglandins of COX origin have been demonstrated to induce ocular inflammation that underlies the pathological progression of diabetic retinopathy, both by itself and in its ability to up-regulate vascular endothelial growth factor (VEGF)[170] [171] [172] [173] [174]. Inflammatory prostanoids are known to be synthesized by COX-2 under elevated glucose conditions[175] [176]. Even in the newly diagnosed diabetic, alterations in hyperpermeability are visible, and considered to be mediated by both VEGF and TNFα[177] [178] [179]. VEGF has an established role in increasing expression of intercellular adhesion molecule-1 (ICAM-1), which exacerbates cytokine secretion via leukocyte activation²¹⁹. ICAM-1 expression also increases in the earliest stages of diabetes due to oxidative stress mediated by hyperglycemia^{169 219}. Increases in ICAM-1 levels in diabetics are related to neural apoptosis during ischemic conditions[180]. As much as a 3-fold elevation in ICAM-1 levels were observed in diabetic retinal tissue compared to controls[181]. VEGF protein levels are increased during hyperglycemia and in this elevated state, increase COX-mediated inflammatory prostacyclin synthesis via induction of STAT3[182] [183] [184] [185]. STAT3 is a common intercellular kinase that is activated by oxidative stress from free radicals, TNFα, and other mediators of inflammation[186]. Cellular damage via oxidative stress mediated upregulation of VEGF is considered one of the primary pathological mechanisms underlying diabetic retinopathy[187] [188] [189] [190]. Reactive oxygen species (ROS) are synthesized via the mitochondrial electron transport chain and NADPH oxidase under hyperglycemic conditions[191] [192]. Another point of self-perpetuating upregulation between diabetic retinal pathological systems involves the p38MAPK protein. p38 phosphorylation is associated with diabetic vascular hyperpermeability, retinal ganglion apoptosis, NMDA mediated neural cell death, and is activated by hyperglycemia, pro-inflammatory cytokines, and VEGF[193] [194] [195] [196] [197]. Thus we see a complete up-regulating system between free radicals, inflammatory cytokines, VEGF, and the pro-inflammatory COX derived metabolites.

Between these pathological systems, VEGF is considered the central mediator of retinopathy[198] [199]. Furthermore, the mechanisms of retinopathy are considered homologous between rat STZ models and human subjects, as both are mediated by VEGF and VEGFR2 upregulation[200] [201]. Rat, mouse, and human models of diabetic retinopathy identify neuronal and glial apoptosis, even in the earliest stages of the disease[202] [203] [204] [205]. In addition, both Type 1 and 2 DM induced retinopathy is characterized by similar biochemical and microvascular alterations¹⁸³.

The progression of diabetic retinopathy can be attributed to 4 main pathological processes^{10 11 [206]}, which include microaneurysms, increased vascular permeability, capillary occlusion, and neovascular proliferation. The blood-retinal barrier, composed of pericytes and endothelial cells, often becomes damaged in the diabetic pro-inflammatory and hyperglycemic state[207]. As pericytes are the main nutritional source for retinal endothelial cells, the blood-retinal barrier’s protective functions become impaired and can lead to capillary leakage[208]. In the more advanced stages, this pathology can give rise to macular edema and complete blindness[209]. Diabetic induced retinal hyperpermeability is directly proportional to the elevation in VEGF expression¹⁷⁷ [210] [211] [212]. VEGF induces NOs activation resulting in prolonged hyperpermeability and down-regulation of the occluding protein, found on tight junctions[213] [214] [215]. Hyperglycemia is known to cause capillary occlusion, which subsequently creates pockets of retinal ischemia and hypoxia[216]. These two conditions set the stage for angiogenesis primarily mediated by VEGF. Neovascularization occurs to return blood flow to ischemic areas[217]. VEGF synthesized by the vascular smooth muscle layer, pigmented epithelium cells, pericytes, and neural retina, as well as its receptor VEGFR2 on epithelial cells, are both upregulated after retinal ischemia¹⁷⁹ [218] [219] [220] [221] [222]. Differentiation and proliferation of endothelial cells is mediated by VEGF, resulting in the formation of new capillaries[223].

VEGF mRNA stability and expression are enhanced under hypoxic conditions[224]. Hyperglycemia not only up-regulates VEGF via hypoxic conditions, but increases VEGF through increasing TNFα, IGF-1, advanced glycation end products (AGEs), ROS, and multiple ILs of which are all elevated in the diabetic condition[225] [226] [227] [228] [229] [230]. The free radicals NO, superoxide anion, and peroxide are quickly produced upon VEGF-VEGFR2 binding and are incorporated into the activation of VEGFs mitogenic cascade^{225 [231] [232] [233]} . As with many of the other mechanisms discussed, the relationship between AGEs, ROS, oxidative stress, and inflammatory regulators in the retina, is an up-regulating system. The polyol pathway is well established in diabetics. Occurring primarily in tissues non-responsive to insulin mediated glucose uptake, exhausted glycolytic enzymes reduce activity, followed by increases in sorbitol and fructose accumulation via the activity of aldose reductase and sorbitol dehydrogenase[234]. Elevated levels of sorbitol and fructose then elevate the NADH/NAD⁺ ratio[235] [236] [237], resulting in a hypoxic condition²³⁵. In addition to oxidative stress, the polyol pathway becomes activated in response to prostaglandin synthesis and COX activity[238] [239] [240]. Hyperglycemia also stimulates phosphorylation of the DAG-PKC pathway[241], whose activation is also increased by an elevation in the NADH/NAD⁺ ratio^{235 [242]} . PKC activation and resulting DAG accumulation are well established as mediators enhancing the secretion of many growth factors involved in angiogensis^{237 241 [243] [244]}. Free radicals in the form of hydroxyl anions accumulate in the diabetic condition as a result of glucose auto oxidation in ketoacidotic environments[245]. We previously mentioned the VEGF induced NO accumulation as a means of increasing hyperpermeability; here we review other mechanisms leading to NO production in both retinopathy and glaucoma. Glutamatergic transmission occurs in photoreceptor, bipolar, and ganglion cells of the retina (Adamis, 1994). Increased glutamate secretion occurs in response to capillary occlusion. Over-activation of the glutamate-NMDA receptor results in an accumulation of intracellular Ca²⁺, which in turn activates the NOs enzyme(Takeda, 2001)[246] [247]. Excess NO production favors synthesis of peroxynitrites from free radical anions, which impart an exponentially greater response of cellular damage[248] than anions alone. Retinal ischemic injury is directly associated with increases in metabolites from NO, superoxides, and peroxynitrite[249]. Above normal physiological concentrations of free radicals can favor phosphorylation of PKC, increase sorbitol levels, translocate NF-κβ, and favor the non-enzymatic formations of AGEs[250]. The non-enzymatic covalent binding of glucose to long-lived proteins and lipids under hyperglycemic conditions creates AGEs[251]. AGEs in turn, are known to promote pro-inflammatory cytokine secretion and NF-κβ activation[252] [253] [254] [255].

This entire up-regulatory pathology of retinopathy can be alleviated by administration of whole-Cannabis plant material. Besides the anti-inflammatory effects of cannabinoids, non-cannabinoid derived products from the marijuana plant have proven successful in reducing COX derived metabolites that are elevated in the diabetic condition. Cannflavin is a prenylated flavone antioxidant unique to the marijuana plant. Cannflavin has 30x more potent of a COX inhibitory effect than aspirin[256] [257]. General constituents of the volatile fraction of a Cannabis extract have demonstrated anti-inflammatory properties via COX inhibition[258] [259]. The carageenan induced paw edema assay is used to study a multitude of inflammatory pathways in rodents. In this design, THC has been tested to have twice the anti-inflammatory strength as hydrocortisone, and more profoundly, 80x the potency of aspirin[260] [261]. Utilizing the same model, other groups identified the cannabinoid CBC to have quite a potent anti-inflammatory characteristic[262] [263]. The pyrrolized metabolites of CBD also demonstrate a substantial decrease in COX-1 activation[264].

Cannabinoids have profound inhibitory effects on angiogenesis via inhibition of the VEGF pathway[265] [266]. CBD inhibits ICAM-1 expression¹⁸¹, thereby reducing the inflammatory cytokine response, neural apoptosis, and retinal ischemia¹⁸⁰ observed in retinopathy. CBD also reduces TNFα in retinal tissue, as determined using a sandwich ELISA¹⁸¹. p38 activation is markedly inhibited following CBD treatment, as well as almost complete inhibition of tyrosine nitration¹⁸¹ . The same group found CBD to also prevent hyperpermeability and cell death. Both in vitro and in vivo studies identify antioxidants as therapeutic agents to prevent glutamatergic/NMDA induced neurotoxicity resultant from ischemia[267] [268]. THC, CBD, and Win55,212 antagonize glutamatergic neurotoxicity via a CB1 dependent mechanism in individual neurons in vitro as well as in whole brain studies[269] [270] [271]. THC, CBD, and the synthetic agonist HU-210 all behave as potent antioxidants, protecting against free radical and glutamatergic cell death[272] [273]. THC not only prevents blood-retinal-barrier degradation, but also restores the damaged tissue to original thickness[274], thereby inhibiting vascular hyperpermeability. Both THC and CBD (.4 & 2mg/kg) inhibit neuronal apoptosis and NMDA mediated tyrosine nitration induced by NMDA (200nmol/eye)²⁷⁴, as determined by thickness of retina in combination with a TUNEL assay. The same research group confirmed these findings using an immunohistochemical slot blot technique in addition to immunofluorescence of nitrated tyrosine residues. The authors went further to identify specifically how THC inhibits destruction of retinal neural conduction. Utilizing RT-PCR, retinal ganglion cell mass was measured with the Thy-1 antigen, and ganglion axon with the NF-L marker[275] [276]. After discovering that elevations in NMDA promoted loss of both Thy-1 and NF-L, the group found that THC inhibits breakdown of both these structures in a dose-dependant fashion. THC and CBD both offer unique therapeutic benefits. CBD increases the stability of the endocannabinoid anandamide, a well documented, potent neuroprotectant acting at a multitude of receptor and non-receptor mediated mechanisms[277] [278] [279] [280]. CB1 activation by THC promotes phosphorylation of neuroprotective MAPK pathways[281]. THC also inhibits production of nitrite and nitrate free radicals in the retina²⁷⁴. In addition to these beneficial effects for both retinopathy and glaucoma, both animal and human studies identify the CB1 agonists THC, Win55,212, 2-AG, and HU-211 to alleviate the intraocular eye pressure associated with hyperpermeability[282] [283] [284] [285] [286].

5. Cannabinoids and Atherosclerosis:

Atherosclerosis is a common disease occurring in diabetics[287] [288]. This is likely due to similar chronic inflammatory and adhesion protein pathological states. Severe clinical complications can occur acutely upon thrombosis and plaque rupture[289] [290]. Elevated levels of ICAM-1, VCAM-1, and E-selectin have been identified in the development of atherosclerosis, Type 1, and Type 2 diabetes mellitus, in addition to being correlated to both hyperinsulinemia and hyperglycemia [291] [292] [293] [294] [295] [296] [297]. Advancement of Atherosclerosis results from imbalances between the Th1/Th2 cytokine profile[298], due to endothelial injury. As with the imbalance observed in diabetes, there is an up-regulation of Th1 inflammatory cytokines seen in early and advanced atherosclerotic lesions[299] [300]. The ApoE-/- mouse model of Atherosclerosis mimics human pathology of this disease[301]. Their hypocholesteremic condition results in fat accumulation within vessel walls, with visible lesions occurring by the 5^th week of induced high fat diet.

In both human and rodents, immunohistochemical techniques reveal the distribution of CB2 receptors within atherosclerotic lesions, whereas no staining occurs within healthy arteries^{298 [302]} . CBD was discovered to block macrophage chemotaxis, an early stage of atherosclerotic lesion formation, in a CB2 dependent fashion, both in vivo and in vitro[303] . Also found to be CB2 dependent was THC’s ability to block leukocyte adhesion in mouse models of atherosclerosis²⁹⁸. THC has been found in vitro to inhibit macrophage chemotaxis resulting from MCP-1 activation, as well as down-regulating chemokine receptor CCR2. Both effects were blocked by the CB2 antagonist SR144528[304] . Both IFNγ of lymphoid origin and macrophage infiltration of developed lesions is decreased with THC treatment ²⁹⁹ . Both the CB2 selective agonist JWH and the endocannabinoid anandamide inhibit CD8 T-lymphocyte chemotaxis resulting from CXCL12 receptor activation[305].

Utilizing the ApoE-/- atherosclerosis model²⁹⁸, as well as in vivo[306] [307], THC, CBD, and anandamide have all been found to attenuate advancement of atherosclerotic lesion formation. Within 11 weeks of a high fat diet in ApoE-/- mice, atherosclerotic lesions are visible throughout aortic roots. THC treatment in this model demonstrated similar arterial composition to controls²⁹⁸. Other beneficial effects of THC in atherosclerotic development that were abolished by SR144528 included reduced macrophage infiltration, leukocyte adhesion, MCP-1 induced leukocyte migration, and decreased TNFα stimulated CCR2 up-regulation.

Attenuation of atherosclerotic pathology of cannabinoids lies once again in their ability to bind to PPARγ. PPARγ agonists have been demonstrated to increase lipid storage while simultaneously inhibit cytokine stimulated macrophage activation[308] [309] [310] [311]. The production of inflammatory mediators in T-cells, endothelial cells, and smooth muscle cells, has been found to be inhibited by PPARγ ligands[312] [313]. Given these beneficial preventative effects of phytocannabinoids, in combination with the ability of THC to shift the T-cell balance towards a Th2/Th1 profile^{165 [314]} in humans, we find ample evidence demonstrating cannabis may have a beneficial effect in diabetics by reducing development and progression of atherosclerotic lesions.

6. Cannabis and Cardiovascular Complications of Diabetes:

At the beginning of this presentation we discussed the high incidence of heart disease and stroke, as well as the mortality rate of diabetics due to hypertension. We had also mentioned conditions of hypoxia due to hyperglycemia. Here we shall now discuss the beneficial effects of marijuana in the diabetic state with regards to cardiovascular complications.

The endocannabinoid anandamide is responsible for vasodilation, mediated at the transient receptor potential vanilloid receptor (TRPV-1)[315]. Importance of cardiovascular effects of endocannabinoids in consideration of phytocannabinoid treatment lies in the discovery that CBD can inhibit both enzymatic degredation by anandamide amidase as well as block anandamide reuptake[316] [317]. Repeated treatments with THC cause down-regulation of sympathetic nervous system activity and increased parasympathetic activity resulting in brachycardia and lowered blood pressure in humans[318]. Animal models also display hypotension and brachycardia[319]. The vasorelaxant effects of THC are mediated by PPARγ[320]. PPARγ activators have been correlated to vasorelaxation, elevated NO bioavailability, blood pressure decreases, and reduced atherosclerotic development[321] [322]. In a rodent model, THC (10μM) and the TZD, rosiglitazone (30μM) followed homologous cardiovascular effects³²⁰. In this study, CB1 antagonists were found ineffective at inhibiting these effects, while a PPARγ inhibitor completely blocked vasodilation. THC was also found to elevate levels of the same prostanoids associated with the vasodilatory effects of TZDs[323] .

The CB1 agonist HU-211 displays an elevated attenuation to epinephrine’s arrythmogenic effects during arterial occlusion and reperfusion in rodent models[324]. Ischemic conditions result from various types of arterial and cerebral occlusions in diabetes resulting from hyperglycemia. Under animal models of ischemia-induced brain damage, therapies inducing hypothermia prove most efficacious[325]. THC has proven to be effective at induction of hypothermia[326]. Both THC and CBD attenuate the oxidative potential during an infarction as assessed by cyclic voltammetry[327]. THC and CBD also decrease the volume of infarction resulting from cerebral capillary occlusion in rodents[328]. Both the hypothermic and infarction effects of these cannabinoids were completely abolished by the CB1 antagonist SR141716.

THC can protect the heart from hypoxia. Using cardiomyocytes under hypoxic conditions with no glucose, THC had a maximal reduction of lactate dehydrogenase release, an indicator of oxidative stress, from 388% to 129% normal concentrations in controls[329]. Interestingly, LDH release was not affected by THC under normal oxygen conditions. The effect was found to be inhibited by the CB2 selective antagonist SR144528. It has long been known the cardioprotective effects of pre-conditioning induced NO production against hypoxic cellular stress[330]. iNOS-mediated NO production has beneficial vasodilatory effects in heart cells in vivo[331] [332]. THC mediated elevations in NO by cardiac cells was found to be CB2 dependent³²². This same group found that hypoxia reduces cardiac fiber density. THC treatments resulted in indistinguishable fibers between THC treated tissues and controls.

It should be clarified that localized production of NO can have both beneficial and negative consequences in diabetes. Various knockout studies identify nervous system localized nNOS and iNOS to increase neural damage caused by cerebral ischemia and arterial occlusion, and eNOS to prevent injury from ischemia[333]. CB1 activation on cerebellar granule cells blocks membrane depolarization and inhibits nNOS Ca²⁺ -dependent NO production[334]. nNOS and CB1 co-localization has been demonstrated with high homology throughout the nervous system with similar results being reported[335]. The vasodilatory effect of anandamide is conferred via NO production by eNOS, as demonstrated by blocking with L-NAME[336]. The soluble adhesion molecules MCP-1, ICAM-1, and VCAM-1, as well as platelet aggregation, are all inhibited by eNOS[337]. Indeed, hyperglycemia has an inhibitory effect on endothelial vasodilation[338] [339]. Additionally, TNFα is known to activate iNOS[340] in the nervous system, leading to such conditions as hyperalgesia.

7. Cannabis, Neuropathies, Excitoxicity, and ROS:

Neurodegeneration is a fundamental process in retinopathy²⁰³. Ganglion and retinal cell death occur from neurotoxicity¹⁸¹. Neural loss and injury have been implicated in diabetes from the brain to the periphery[341]. Other neurologic complications of diabetes observed in humans and rodents include dementia, learning deficits, Alzheimer’s, decreased abilities on neuropsychological tests, and an extremely high incidence of depression[342] [343] [344] [345] [346] [347] [348] [349] [350] [351].

Glutamatergic excitotoxicity is a well documented pathological state leading to various neuropathies in diabetes[352]. Specifically, hyperglycemic-induced ischemia activates glutamatergic-excitotoxic apoptosis[353] [354] [355] [356]. Neurotoxicity via glutamatergic signaling occurs through several mechanisms at the NMDA receptor. Associated with excessive activation of the NMDA receptor is an elevation in intracellular Ca²⁺, which subsequently generates mitochondrial and apoptotic reactive oxygen species (ROS)[357] [358]. Apoptotic events are associated with diabetic neurological, retinal, endothelial, and kidney complications^{247 [359] [360]}. Nervous system cells known to be damaged and signaled to apoptosis under hyperosmolar conditions include Schwann cells, neuroblastoma, dorsal root ganglion, and hippocampal neural circuits[361] [362]. Apoptosis is also a fundamental phenomenon occurring in STZ induced experimental rodent models of diabetes[363] [364] [365]. Cell death can be initiated by increases in intracellular Ca²⁺, which in turn signal cytoskeletal degredation, impaired energy expenditure, and activate hydrolytic enzymes[366]. Inflammatory mediators are also known to play a pivotal role in diabetic neurodegenerative pathology. ERK, JNK, and p38 have all been found to be elevated during NO and NMDA induced apoptotic events[367] [368] [369] [370]. Axonal degeneration is a well established feature of increased JNK phosphorylation[371] [372]. IGF-1 has been shown to exert an anti-inflammatory/neuroprotective, bi-directional regulatory control over JKK and p38 phosphorylation states[373] [374] [375] [376] [377]. These studies additionally identify oxidative stress as the means by which NF-Kβ becomes activated and leads to axonal death. Improper IGF-1 signaling and decreased levels have been identified in Type 1 diabetics. Neuronal damage has been linked to up-regulation of nNOS activity and COX metabolites[378]. Hyperglycemia itself directly causes increased superoxide production, mitochondrial dysregulation, and stresses of oxidative and nitrosative origin to the nervous system[379].

Further means of neural degeneration in diabetes occur by decreased neurotrophic and antioxidant activities[380] [381] [382]. The anti-apoptotic and neuroprotective effects of both C-reactive peptide (CRP) and insulin are well characterized[383] [384]. CRP treatment benefits patients by enhancing autonomic nerve conduction and metabolism, as well as increasing neuro-regeneration[385] [386]. Similar to cannabinoids, CRP activates eNOS, thereby enhancing vascular flow[387]. Neurotrophic growth factor (NGF) is impaired in STZ diabetes induced rodents and exerts trophic signaling for small-sensory neuron homeostasis[388] [389] [390] [391]. Bradykinin and H-ion potentiated inflammatory nociception is further enhanced by NGF, in addition to up-regulating secretion of hyperalgesic neurotransmitters[392] [393] [394] [395]. Taurine functions as an osmolyte, neurotrophic factor, and antioxidant; its levels have been shown to be decreased in the diabetic state[396] [397]. Hyperglycemia elevates fructose and sorbitol intracellular concentrations resulting in depletion of osmolytes, including taurine[398].

The results of these various oxidative and inflammatory attacks on the nervous system reflect the unique types of analgesia seen in the diabetic state in addition to diabetic neuropathies. Two common symptoms of diabetes include thermal hyperalgesia and mechanical allodynia[399] [400] [401] [402] [403], both in humans and rodent models. Diabetic neuropathies are often one of the most difficult forms of analgesia to treat[404] [405] [406] [407]. Opiates prove limited in efficacy under both clinical trials and experimental animal models[408] [409] [410] [411].

Neuropathic pain has been found to be mediated by TNFα, IL-1, and IL-6[412]. TNFα can both activate nociceptive transmission and induce hyperalgesia[413] [414]. Hyperalgesia was decreased by the inhibition of TNFα within the dorsal root ganglia, and was found to be mediated by blocking p38 activation[415]. NGF is known to potentiate nociception and is up-regulated by IL-1β[416]. Macrophage-mediated Schwann cell denervation is mediated via MCP-1[417]. Utilizing knockout mice for the MCP-1 receptor CCR2, mechanical hyperalgesia does not develop after a partial nerve ligation[418] [419]. Numerous studies have demonstrated allodynia in diabetes to be associated with Aβ and Aδ malfunction in sensory input[420] [421].

Cannabinoid therapy not only alleviates the sensory complications of diabetic neuropathy, but can prevent its development. Numerous studies have identified phytocannabinoids, CB1, and CB2 as successful therapeutics and targets in treating neuropathic mechanical allodynia and thermal hyperalgesia[422] [423] [424] [425] [426] [427] [428] [429] [430] [431] [432] [433] [434] [435] . Most of these studies have been conducted in animal models. Human and rodent neuropathies are identical in tactile features, as nerve injury induces homologous allodynic and hyperalgesic effects[436]. Furthermore, rodent models are considered more quantifiable in terms of hyperalgesia and effectiveness of treatment[437]. Nevertheless, clinical trial data has now been published that demonstrates a statistically significant reduction in neuropathic pain with the use of smoked marijuana [438] .

In addition to neural synthesis of NGF, mast cells have been shown to secrete this factor as well[439]. NGF can also stimulate the secretion of numerous inflammatory proteins of mast cell origin, including itself, thus forming self up-regulation[440] [441] [442] [443] [444]. Nociceptive signaling via NGF and its receptor trkA is attenuated via cannabinoid application^{440 441}. Numerous primary afferent neurons express both CB1 and secrete NGF[445] [446]. Both trkA and CB2 are located on the mast cell membrane[447], and the endocannabinoids PEA and AEA have been found to block trkA expression and prevent NGF mediated hyperalgesia[448] [449].

We previously mentioned the association between allodynia and dysregulation of Aβ and Aδ inputs. CB1 expression displays an extremely high density within both these nociceptive fiber types[450] [451] [452] . Upon nerve injury within the periphery, numerous anatomical and immunohistochemical procedures identify an up-regulation of both CB1 and CB2[453] [454] [455]. Utilizing the CB1 antagonist SR141716A, mechanical allodynia and thermal hyperalgesia are increased in rodent models⁴²³. Cannabinoids inhibit neuropathic algesia via a similar mechanism to the most widely prescribed drug for the treatment of neuropathy, gabapentin[456] [457]. Gabapentin, and other drugs commonly used to treat neuropathy, have all proven unsatisfactory in efficacy or are correlated to numerous harmful side effects[458] [459] [460] [461]. Gabapentin reduces hyperalgesia via the inhibition of voltage-gated calcium channels of the L-, R-, P/Q-, I-, and N-types and subsequent intracellular reduction of Ca^{2+ [462] [463]}. Cannabinoids acting at the CB1 receptor have been demonstrated to inhibit Ca²⁺ currents in N-, P/Q-, and L- channel types[464] [465] [466] [467].

In terms of preventing or blocking the mechanisms of neuropathy, cannabinoids act via multiple antioxidant and antiexcitotoxic pathways, thereby not only treating symptomology, but facilitating normal physiologic function in diabetic pathology. Both NMDA and β-amyloid neurotoxicity are attenuated by CBD[468]. It is a generally accepted trend that CB1 agonists are neuroprotective from ischemic and excitotoxic events^{269 273} [469] [470] [471] . The endogenous cannabinoid system is up-regulated during hypoxia and protects cells from oxidative damage²⁷³. These neuroprotective effects are also evident by the use of CB1 antagonists such as SR171416A, also known as Rimonabant. Under a model of NMDA induced neurotoxicity, the CB1 agonist Win55,212 reduced toxicity by 65%, an effect that was completely abolished by Rimonabant[472]. This same study found NMDA toxicity to be nNOS dependent, as activity at the NMDA receptor increased fluorescence of an NO tag by 160%. Win55,212 completely abolished NO production, an effect that was blocked by Rimonabant.

Cannabinoids prevent the formation of ROS and exert cellular protective effects via multiple mechanisms that are of benefit to diabetics. Neuroprotection is mediated via a PI3K/AKT dependent mechanism initiated by CB1 agonists, resulting in decreased p38 phosphorylation[473]. THC causes a decrease in p38 phosphorylation and a resulting inhibition in ROS formation and apoptosis[474]. Mitochondrial superoxide overproduction occurs in states of hyperglycemia²⁵⁰. The hexosamine pathway has been demonstrated to become activated by superoxides[475]. eNOS activity is inhibited by both superoxides and metabolites of the hexosamine pathway³³⁷. The extremely high concentration of ROS in diabetics results from four major pathways, including the polyol, hexosamine, PKC, and advanced glycation end products (AGEs)[476]. NF-κβ activation has been correlated to all 4 pathways in addition to promoting endothelial leukocyte adhesion and up-regulation of Th1 inflammatory cytokines^{237 476} [477] [478]. We previously discussed the cannabinoid mediated increase in eNOs activity, in addition to lowering the Th1 cytokine profile.

Ischemic-mediated production of ROS and subsequent cellular injury is found to be FeCl2 dependent[479] [480]. Powerful oxides are formed from the release of Fe2+ via a Fenton reaction with H2O2. Using lactate dehydrogenase (LDH) as a marker of cortical neuronal apoptosis, FeCl2 within ischemic concentrations can induce as much as a 70% release of LDH from neurons[481]. When testing various cannabinoid receptor agonists under a model of FeCl2 induced neurotoxicity, apoptosis is reduced by as much as 50%⁴⁸¹. The same study identified significant reductions in fluorescent detection of the oxidative product ethidium. Utilizing various antagonists and inhibitors, this research group identified the molecular mechanism of ROS inhibition by cannabinoids to be via inhibition of cAMP accumulation and subsequent PKA activation. Both cAMP and PKA have been implicated in the formation of ROS in neural and epithelial cells under states of excess activation[482]. Numerous cell lines are known to produce ROS from a PKA dependent mechanism including leptin activated epithelial cells, cardiomyocytes, and fibrosarcomas[483] [484] [485]. The CB1 receptor is well known for its ability to inhibit cAMP production and its stimulating effect on PKA phosphorylation[486]. Using the PKA activator dbcAMP or the CB1 antagonist Rimonabant, the neuroprotectant effect of CB1 agonists was completely abolished. PKA dependent ROS formation and oxidative damage from H202 and BSO were also found to be inhibited by CB1 agonists⁴⁸¹. Further support for the CB1 receptor’s role in ischemic damage lies in the discovery that it and the endogenous cannabinoid PEA are up-regulated after hypoxia and reduce the resulting inflammatory response[487] [488].

We have discussed several neuroprotective effects of cannabinoid agonists that are receptor mediated, but potent antioxidant effects are exerted via non-receptor mediated mechanisms[489]. Overproduction of superoxides is known to occur in diabetic humans, in nearly every tissue type, including the retina, kidney, endothelium, nervous, and cardiovascular system^{476 237}. Oxidative stress in diabetics can be evaluated via the degree of lipid peroxidation, of which, is typically elevated compared to non-diabetics[490]. In rodent models of neuropathy, prostaglandin E2 (PGE2) concentration is more than doubled, associated with this elevation is an increase in both lipid peroxidation and subsequent ROS[491].

The New York Academy of Sciences officially recognizes the potency of cannabinoids as antioxidants, quoting “ In a head to head trail of abilities of various antioxidants to prevent glutamate toxicity, cannabidiol was superior to both α-tocopherol an ascorbate in protective capacity”²⁷². Specifically, this study found CBD to be 50% more potent an antioxidant than ascorbate. Utilizing numerous models, the Academy of Science determined that CBD can prevent H2O2 induced apoptosis by 75%. CBD has been found to be safe at such extremely high doses as 10mg/kg/day in human clinical trails with limited to no side effects[492]. This has a far safer therapeutic potential than even the over-the-counter antioxidant BHT, which even in small quantities has been linked to tumor formation[493] [494].

Ischemic conditions result in excessive glutamatergic release creating neurotoxicity by overactivation of NMDA, kainate, or AMPA receptors by elevating intracellular Ca²⁺ to toxic levels[495]. Glutamatergic excitotoxicity is also ROS dependent^{267 268}. These forms of toxicity have proven to be diminished with the administration of antioxidants both in vitro and in vivo^{267 268}. Specifically, ischemic mediated ROS production can be alleviated by antioxidants such as α-tocopherol. Antioxidants can be of various structural forms as their distinctive feature is the ability to oxidize with ease. Ascorbate and tocopherols are among the best known. Besides the ability to oxidize, antioxidants have been demonstrated to inhibit iNOS and COX-2 transcription[496] [497]. Cyclic voltammetry may not be an in vivo model, but its results can be extrapolated to such instances due to the quantitated measurement of the ability of the compound to donate or accept electrons. In yet another Ney York Academy of Sciences publication²⁷², cyclic voltammetry was used to assess the antioxidant potential of several vitamin antioxidants, BHT, THC, CBD, CBN, and HU-210. It was discovered that all the phytocannabinoids had equal or greater antioxidant potential than BHT. Under numerous cell models and protocols used to study antioxidant properties against ROS products of Fenton reactions, both THC and CBD were comparable or greater in efficacy to BHT. When comparing ascorbate, α-tocopherol, BHT, CBD, and THC in antioxidant effects against AMPA and kainite receptor excitotoxicity, CBD was found to have far superior properties than all others tested.

Cannabis may also exert antioxidant properties against the formation of AGEs. Marijuana from numerous landraces, cultivars, and hybrids have been found to contain significant concentrations of flavanoids, including quercitin and kaempferol[498]. As little as .5-10μg of quercitin or kaempferol can reduce hemoglobin glycosylation by as much as 52% and 15%, respectively[499].

8. Marijuana, Diabetes, and Depression:

A previous petition was submitted to include anxiety and depression on Amendment 20 and was denied due in part to an inability to link specific subtypes of depression to a specific mechanism of cannabinoid efficacy in treatment. Here we provide additional evidence to directly link at least one form of depression to benefits from marijuana.

New molecular evidence demonstrates a link between homologous modes of action in cannabinoid and Fluoxetine antidepressant efficacy. At the beginning of this paper and the discussion on neurological complications, we cite numerous in vivo, in vitro, and clinical studies demonstrating a high incidence of depression in diabetics. In animal models of type 1 diabetes, numerous complications are reported in the hippocampus, cerebral cortex, hypothalamus, and overall limbic system, including glutamatergic neurotoxicity, hippocampal cell death, decreased neurogenesis, and lowered synaptic plasticity[500] [501] [502] [503] [504]. Much of this pathology can be correlated to oxidative stress[505].

Human Type 1 diabetics have been demonstrated to display decreased hippocampal neurogenesis[506]. Interestingly, in a clinical trial treatment with Fluoxetine not only reduced depressive symptoms, but significantly brought glycemic values back into control[507]. As we have previously discussed the implications of cannabinoid neural stem/ progenitor cell neurogenesis within the hippocampus, reductions in cAMP, homology in mechanism to benzodiazepines and over a dozen antidepressants, we will not discuss the antidepressant effects of cannabinoids in detail here, but resubmit the depression petition as supporting evidence for this section. New evidence has been gathered however, that directly implicates efficacy of antidepressant drugs for various mood disorders and their mode of therapeutic efficacy being mediated by neurogenesis within the limbic structures[508] [509] [510] [511] [512] [513] [514] [515] [516].

9. Important Synergistic Interactions

It is noteworthy to mention that interactions between exogenous and endogenous cannabinoids can create a potent synergistic activity in numerous pathways for diabetic complications. Anandamide hydrolysis can be inhibited by CBD administration³¹⁷. CBD also inhibits the metabolism of THC into 11-hydroxy THC, thus mediating a reduction in the psychoactive properties of the plant, as this THC metabolite displays significantly more potent psychological effects³⁰⁶. Thus all previously mentioned therapeutic effects of anandamide can be considered of benefit to the diabetic patient, as its influences are potentiated by phytocannabinoids.

10. Marijuana, Hyperglycemia, Hyperinsulinemia, β-Cell Regulation, and Insulin Signaling:

Numerous human clinical trials and animal models both find hyperglycemia to be an independent risk factor for the various microangiopathies correlated to diabetes[517] [518] [519] [520]. The pathogenic progression of T2D can be correlated to hyperinsulinemia, often the first detectable complication of the disease[521] [522] [523] [524] [525] [526] [527]. Abundant data has accumulated demonstrating the importance of insulin hypersecretion in the pathogenic progression of T2D[528] [529] [530] [531] [532] [533] [534] [535] . Both insulin secretion and sensitivity are affected in the hyperinsulinemic state[536] [537]. Hyperinsulinemia precedes the onset of T2D[538] [539] [540].

Hyperglycemia is also responsible for insulin resistance, in addition to its contributions to all the various complications associated with both forms of diabetes mellitus. As a coping mechanism, T-lymphocytes develop insulin receptors under hyperglycemic conditions, with concomitant lipid peroxidation and resulting ROS production[541] [542] [543] [544] [545] [546]. Diabetic patients in ketoacidosis display significantly higher levels of TNFα, IL-1β, IL-1βR, IL-8, and CRP[547]. The AKT signaling pathway activates eNOS^{237 427}. Hyperglycemia inhibits eNOS activity via hexosamine metabolites direct O-linked glycosylated modifications to the AKT protein³³⁷.

Insulin secretion occurs in a pulsatile manner within the β-cell, with the opening of VGCCs allowing an intracellular accumulation of Ca^{2+ [548] [549] [550]} . VGCC closure and subsequent inhibition of intracellular Ca²⁺ levels is a well documented feature associated with agonist activity at both CB receptors^{464 465 [551] [552] [553]} .

As we shall demonstrate, marijuana may reduce the harmful effects of hyperinsulinemia, increase glucose metabolism, and enhance insulin signaling. Both cannabinoid receptors have been identified within islet cells particularly CB1 being predominant on α-cells while CB2 is localized to both α- and β-cells[554]. CB2 agonists such as anandamide, 2-AG, methanandamide, and JWH have been demonstrated to reduce glucose-evoked insulin secretion by as much as 30%⁵⁵⁴. Effects were abolished by administration of the CB2 antagonist AM630.

At first consideration one might view this effect as harmful, however, the additional metabolic activities of cannabinoids in conjunction with decreased insulin secretion gives an overall benefit by enhancing glucose uptake without requiring additional insulin. Glucose uptake increases by as much as 160% in 3T3-L1 adipocytes pretreated with AEA (anandamide)[555]. The CB1 selective agonist ACEA enhances glucose uptake in human endothelial cells[556]. In vivo stimulation of glucose uptake in numerous tissues, including skeletal muscle, adipose, and endothelial, is NOS dependent[557]. Anandamide, and other CB1 agonists previously discussed in the NOS section can increase NOS in these tissues[558].

Arachidonic acid (AA) enhances both basal and insulin stimulated glucose catabolism while COX-2 synthesized products of AA inhibit glucose metabolism[559] [560] [561]. We had previously discussed the cannabinoid mediated benefits of COX-2 inhibition.

Activation of the PI3K and AKT/PKB signaling pathways is a well documented phenomenon associated with CB receptor signaling[562] [563] [564]. Specifically, Rimonabant has been demonstrated to inhibit the IR stimulated activation of PI3K via ERK⁵⁶⁴. This demonstrates unequivocally at the exact phosphorylation sites and signaling proteins that cannabinoids have a homologous signaling pathway of medical benefit to the IR signaling pathway. Signaling pathways of both the CB1 and insulin receptors converge at ERK phosphorylation[565]. Activation of either CB1 or the IR causes phosphorylation of PI3K_1b, an effect which is blocked from both signaling pathways by the PI3K_1b inhibitor Wortmannin⁵⁶⁴. Additionally, numerous studies show support for a CB1 mediated phosphorylation of ERK[566] [567] [568]. As the PI3K/AKT pathway is responsible for GLUT-4 translocation, in addition to numerous other metabolically beneficial actions resulting from the anti-apoptotic signals of ERK, we find that CB activation can enhance the effects of insulin signaling while CB2 activation results in reduced insulin secretion.

11. Personal Testimony/ Anecdotal Evidence:

As a 23 year diabetic with severe gastroparesis and sensory neuropathy, I know all to well the pain, nausea, and debilitations my disease has imposed upon me. I began my college career in 2000, only to withdraw a year later due to an inability to drive, a constant need for a bathroom, and general feelings of discomfort (tactile sensitivity). As a once recreational user of cannabis, I used the drug on occasion. On one such event, my recreational use coincided with a day of extreme pain and vomiting. In less than 5 minutes of inhalation, my nausea went away completely, and my pain became more of a minor pressure than burning sensation. Since then I have used medical marijuana under the guidance of my doctor. With repeated, consistent use at regularly scheduled times I also noticed the effects that marijuana had on my blood-glucose levels. I found that smoking marijuana lowered my glucose levels, so much that I began lowering my insulin dosage for the first time in years.

Diabetic ketoacidosis is a truly horrific feeling throughout the body. I often describe it as liquid mosquito bites pumping throughout my veins. Even with consistent Glycohemoglobin A1C levels between 6-7, hyperglycemia affects me 1-2x weekly. Depending on the severity, I may be bedridden for up to 2 days with flu-like symptoms. Smoking marijuana after ketoacidosis dramatically reduces my symptoms, not completely abolishing, but reducing enough such that I may be productive again in a few hours.

There are also times I must eat food due to hypoglycemia, but vomit the food up due to gastroparesis. Cannabis allows me to both hold food down and it stimulates hunger such that I can eat when necessary.

I have a fiancée who loves me unconditionally. I am often frustrated and ashamed because of a frequency with premature ejaculation and impotence. At first this created problems in our relationship, mostly with my own anger and depression with my personal inadequacies. On nights when I must medicate for nausea or pain related reasons, I sometimes find that marijuana helps me to maintain an erection longer.

I do not feel that marijuana is a perfect therapy for diabetes. One problem I find is that I must be disciplined not to eat carbohydrates after medicating: a phenomenon known all to well as the munchies. This emphasizes the importance of marijuana as a medicine and not a recreational drug. A recreational user would act on “the munchies”, whereas a medical patient would be receiving A1Cs and complete bloodwork from their endocrinologist to identify how well they are managing the use of their medicine and diet.

12. Summary:

Diabetes is a debilitating condition due to the numerous pathologies and diseases it predisposes the patient to as a result of its progression. Diabetes is also considerd the 305^th largest cause of death in the United States. Death can be considered the ultimate form of debilitation, thus if cannabis can prevent those complications which contribute to the death of diabetics, the treatment should be considered. The American Diabetes Association, World Health Organization, and numerous studies find an overwhelming incidence of neurologic and metabolic disorders arising in this population. Virtually all diabetics face the fact that neuropathy, blindness, pain, nausea, improper digestion, depression, gastroparesis, sexual dysfunction, and cardiovascular diseases are all possible and highly probable developments in diabetes mellitus. Furthermore, the ADA believes these pathologies to occur similiarly in both Types 1 and 2 of diabetes mellitus, with a quicker progression more evident in Type 1 than 2.

Diabetes is clearly a debilitating and diagnosable disease. A simple glucose tolerance test can determine if a patient is diabetic; a simple ELISA test for CRP or insulin can differentiate between the two subtypes. Although there has been a recent discussion on updating the cut-off values of blood glucose levels in determining a diabetic from a non-diabetic, these values do not vary drastically from the still upheld values determined in the 1980s.

We have discussed numerous metabolic, inflammatory, neurologic, retinal, and free radical pathways resulting in numerous pathologies to various tissues in the diabetic state. Focusing on the molecules as groups in lieu of their mechanisms and tissues, we find:

1. Inflammatory proteins are produced from an up-regulation of Th1. They can also come from adipose tissue, macrophages, neurons, and COX enzymes. Cannabinoids balance the Th1/Th2 balance either towards equilibrium or towards a Th2/Th1 ratio. Numerous cannabinoids were demonstrated to inhibit TNFα, IL-1β, IL-6, as well as benefits against JAK-STAT, JNK, and SOCS protein signaling. We identified how these proteins inhibit numerous activities associated with the insulin receptor and included specific molecular mechanisms of insulin resistance associated with these actions.
2. We also identified how FFAs share similar signaling pathways to these inflammatory proteins, in addition to direct actions of FFAs increasing transcription and/or translation of inflammatory cytokines, thus proving a cyclic upregulation to the pathology. With numerous citations, we demonstrate that cannabinoids work similar to TZDs in reducing insulin resistance but concomitantly increasing adiposity. Marijuana increases weight similar to TZDs; reducing blood glucose concentration via adipocyte lipogenesis.
3. Hyperglycemia favors AA metabolism via COX-2 in numerous tissues, particularly the retina. These inflammatory mediators are associated with increase in free radicals, VEGF, and numerous retinal pathologies. We identified how marijuana products, both of cannabinoid and non-cannabinoid structure can inhibit COX-2 activity, reduce the specific free radicals associated with diabetic retinopathy, and inhibit angiogenesis via down-regulation of VEGF.
4. Hyperglycemia also causes systemic up-regulation of pro-inflammatory cytokines and increases the concentration of numerous free radicals of multiple pathways. Again, we identify how marijuana reduces COX, PKA, excitotoxic, NMDA, hexosamine, NF-κβ, interleukin, TNFα originated mechanisms of free radical production. These free radicals are associated with many of the neurodegenerative effects viewed in diabetics. Additionally, by preventing these free radicals, cannabis helps protect the diabetic from the 3 common microangiopathies: neuropathy, retinopathy, and nephropathy.
5. Hyperglycemia causes numerous cytokines and immune-derived adhesion molecules to become up-regulated, causing the dramatically high incidence of atherosclerosis in diabetics. Here we identified that marijuana inhibits MCP-1 and several adhesion molecules. CB1 and CB2 receptors have been found to become up-regulated on atherosclerotic plaques, agonists are found to decrease progression of plaque formation.
6. Diabetics are far more likely to die from hypertension related complications than the average individual. In line with preventing atherosclerosis via inducing hypertension, the synergistic activities between endo- and exogenous cannabinoids results in increased cardiac output, vasodilation, and increased heart rate with a drop in BP. Every one of these actions are beneficial to diabetic cardiovascular physiology, when combined as a whole.
7. We identified the unique activites of cannabinoids on NOS activities. Interestingly, marijuana constituents have the ability to activate eNOS, increase iNOS in the skeletal muscle and endothelial tissue, and decrease iNOS and nNOS in the nervous and immune systems. Thus cannabinoids help prevent nNOS directed neurodegeneration and peripheral iNOS mediated increases in BP, while increasing glucose metabolism in the skeletal muscle and endothelial tissues.
8. We identified a depletion of naturally occurring antioxidants in diabetics in addition to free radical based damages of every major organ system in the human body. Cyclic voltammetry identifies both THC and CBD as at least 20x more potent of antioxidants than α-tocopherol or ascorbate.
9. We identified specific interrelationships between signaling pathways of the CB and insulin receptors. Cannabinoids can decrease hyperinsulinemia while simultaneously increasing glucose metabolism in numerous tissues.

13. Conclusion:

In conclusion, we find overwhelming evidence to support that marijuana may have a beneficial effect in the treatment of diabetes mellitus of both Type 1 and 2. Due to its schedule I classification clinical trials with marijuana are nearly impossible to perform legally, and the request for human clinical trials by Mr Cologne of the department of health is unacceptable and impossible to fulfill. Here, we utilize numerous studies of in vitro and in vivo to demonstrate a vast multitude of strongly supported mechanisms of therapeutic benefit to the diabetic based on a strong foundation of peer-reviewed support from the literature.

When deciding proper vocabulary to utilize in a legal statute, nonetheless a constitutional amendment, choosing the word “may” implies a significant and substantial room for discussion. If Amendment 20 used the word “must”, this would imply unequivocal, double blind clinical trial, peer reviewed work on large sample populations. “May” is a far broader definition than “must”, and as the Colorado Constitutional Amendment 20 uses the word “may”, review of any petition being submitted under the context of Amendment 20 “must” be reviewed in this broader context, less infringement of Constitutional Rights be the discussion of this petitions review by a judiciary committee.

We thank the Colorado Department of Public Health and Environment for their time and efforts in review of this petition.

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