August 2000 -- Obesity-news

Could obesity be caused by a virus? Over the past few years a team at the University of Wisconsin has been researching a virus found almost exclusively in obese human beings. Although only a few hundred people have been tested, the evidence is overwhelming . . . or it it? Of the subjects who have been examined so far, almost no normal weight individuals have antibodies for the virus, whereas between 15 and 60 percent of obese patients test positive. But one question remains. Does obesity predispose patients to the virus, or does the virus cause the weight gain?

Until a few years ago the idea that a virus could cause obesity would have been laughed into oblivion, but with research pointing to viruses as the cause of other ailments, like ulcers and arthritis, the idea of an obesity virus has been met with interest . . . if not also some skepticism.

No one is suggesting that this virus is the only cause of weight gain, but rather that it may be an additional factor, and an explanation as to why obesity rates are climbing even though our eating and exercise habits have not varied that much over the past decade.

In this month's Obesity-news we look at both the published and unpublished research to bring you an overview of this controversial theory, and comments from the lead researcher, Nikhil Dhurandhar.

In other coverage we bring you an encouraging report on the use of sibutramine in controlled hypertensives, the latest research showing that UCP-3 may be the protein responsible for thermogenesis in humans, as well as an update on the latest drug development advances.

Finally, in our effort to keep you up-to-date on the phentermine shortage situation, we have some additions to our list of phentermine distributors carried in the August supplement. Until the shortage is over, we will bring you information as it becomes available, and will publish regular updates in future editions of Obesity-news.

Does this describe you? Extremely overweight with low cholesterol and triglycerides? If it does, it means you may have been infected by a virus that produces cold symptoms, like sneezing and chills . . . and weight gain. The good news is that if you have antibodies to the virus, known as Ad-36, you are more likely to be successful losing weight and will respond better to weight loss medications. The bad news is no one knows for sure how the virus infects humans, or even if it does. While human subjects have tested positive for the Ad-36 antibody, no one has ever been found with an active case.

Discovering the connection between Ad-36 and human obesity has been a major research project for Drs. Nikhil Dhurandhar and Richard L. Atkinson over the past several years. Dr. Dhurandhar's interest in the possibility that a virus could be a cause of weight gain, goes back to the late 1980s and his days as a physician specializing in obesity in Bombay, India where a similar virus, SMAM-1, was killing hundreds of thousands of chickens.

Obesity virus victims have high weight but low cholesterol.

Animals consumed by a virus should be underweight, not fat. But when the dead birds were examined, it was found that they had a large amount of body fat and low cholesterol, puzzling veterinary pathologist, Sharad Ajinkya, a family friend of Dhurandhar's. The findings also made Dr. Dhurandhar wonder whether there was a connection between the chicken epidemic and the obesity in his patients. So he took blood samples from 52 of them and found that 10 of those patients tested positive for SMAM-1 antibodies. Those 10 were heavier than the other 42 patients and also had lower cholesterol levels, like the chickens killed in the epidemic. 1

Dhurandhar wanted to pursue his findings, but he realized that neither the financing nor facilities were available in India. After closing up his practice and heading for the United States, he interested Dr. Richard Atkinson, an obesity researcher at the University of Wisconsin, in his theory. But they soon ran into a major road block. The US Department of Agriculture would not allow them to import the SMAM-1 virus, Their only choices were to abandon the research entirely, or look for a similar virus. So Atkinson and Dhurandhar scanned a virus database and hit pay dirt, the virus, Ad-36, which had been discovered in the feces of a young German girl with diarrhea. The ease with which they found a new virus also led them to believe that there could be many other such viruses causing weight gain in humans.

To show the connection between Ad-36 and obesity, Dhurandhar and Atkinson have tested hundreds of human subjects, and performed experiments on a wide variety of animals, including chickens, mice and primates. Studies have been published in the International Journal of Obesity and Obesity Research, and several others have been presented at medical conferences.

Studies on AD-36 and obesity.

Animal studies. Chickens. Now that a candidate virus had been found, Dhurandhar needed to determine whether the Ad-36 would produce obesity in chickens as SMAM-1 had in India.

When he inoculated one-day old broilers with the virus they gained more weight and body fat than controls, as well as a second group of chickens inoculated with another avian virus called CELO (chick embryo lethal orphan virus). Like the chickens infected with SMAM-1, those infected with Ad-36 had increased visceral fat, lower serum cholesterol and triglycerides, even though they didn't eat any more than the other birds. 2, 3

Mice and primates. Dhurandhar next looked at whether Ad-36 could produce obesity in mammals, inoculating 35 mice with the virus. When the mice responded similarly to the chickens, he tested mammals more similar to humans — marmosets and monkeys. In the marmoset experiment, 2 animals that tested positive for the virus gained significantly more weight than controls (42.8 ± 6.5 gm versus 14.4 ± 5.9 gm). The infected marmosets also had substantially lower cholesterol, although triglycerides were not affected. 4, 5

Human studies. Bridging the gap to prove that AD-36 could infect humans as well as primates posed a problem. Dhurandhar cold not ethically infect human subjects with an active virus. So he did the next best thing. He tested human subjects for antibodies for the Ad-36 virus, as he had done for SMAM-1 in India. Eventually Dhurandhar and Atkinson would test over 400 subjects in three cities to get the data they needed. Of the first 154 obese volunteers and 45 lean controls, fifteen percent of the obese subjects tested positive, but none of the lean subjects. 6

How does Ad-36 cause obesity?

Although the mechanism by which Ad-36 causes obesity is unclear, it initially seemed reasonable to hypothesize that hypothalamic damage might be a cause. Animals with distemper and borna disease develop brain lesions, and another obesity virus affecting animals, called CVD, had also been shown to cause hypothalamic damage in infected animals. But this did not turn out to be the case with Ad-36. When Dhurandhar examined the brains of Ad-36 infected chickens, he found no damage to the hypothalamus or in any other locations of the brain, eliminating the possibility that a central mechanism was responsible. 2

So Dhurandhar turned his attentions to a more likely culprit, the fat cells themselves. Ad-36 infected animals had larger fat cells, more of them, and a paradoxical reduction of serum cholesterol and triglycerides. When he added Ad-36 to cultures of mouse preadipocytes, he found that they differentiated into mature fat cells at a three times greater rate than preadipocytes from uninfected cells. Dhurandhar also tested Ad-36 cells against adipocytes infected with Ad-2, a virus which is not associated with obesity. After treatment with a differentiation media (MDI), preadipocytes from infected cells developed into mature fat cells as a faster rate than the Ad-2 cells. The triglyceride content of Ad-36 adipocytes was also substantially higher than those of normals cells (27.1 ± 13.6 mg/dL vs. 8.6 ± 4.3), explaining the lower triglyceride levels in Ad-36 infected animals and humans with antibodies to the virus. 9

Weight loss and the Ad-36 antibody.

Since obesity "caught" through the virus is not genetic, it would be expected that individuals who became obese as a result of Ad-36 would have an easier time losing weight than individuals who have a genetic predisposition to obesity. This did turn out to be the case for a group of male patients from the original 154 who participated in a weight reduction program. Ad-36 males lost significantly more weight than obese patients without the virus (17.8% vs. 11.1%). However, this was not the case with female patients. The reason for the gender gap is unknown. It could be that the women also had a genetic predisposition to obesity or that the effect of Ad-36 is different in women. But more testing will need to be conducted before any conclusions can be made.3

With evidence mounting for viruses causing other ailments, such as ulcers and rheumatoid arthritis, the idea of an obesity causing virus is met with less skepticism than it once was. Nonetheless, many in the scientific community are wary, and Dr. Dhurandhar has had difficulty getting his research published. While the chicken and mouse studies were recently reported in the International Journal of Obesity, another journal turned down a manuscript with the human data.

Chicken or the egg: Which comes first the obesity or the virus?

It would be convenient to dismiss the obesity virus as just another ailment caught by fat people, who already have somewhat suppressed immune systems, but Dr. Dhurandhar told Obesity-news that he is convinced this is not the case. In addition to Ad-36, all human subjects were screened for three other adenoviruses—Ad-2, Ad-31 and Ad-37—which are not associated with obesity. Antibodies to these viruses were found equally in lean and obese people, and none was associated with low cholesterol or triglycerides. On the other hand, antibodies to Ad-36 were seen almost exclusively in fat people. This growing body of evidence would certainly lead one to believe that if you catch Ad-36, you are more than likely to wind up gaining weight from it.

Dhurandhar also believes that Ad-36 is only the tip of the iceberg. No major scan of viruses has been conducted, and already several are known to cause obesity in animals, and two in humans — making it likely that others will be found. Further research will need to be conducted to find additional viruses, and scan subjects for antibodies — the ultimate goal being to produce a vaccine to protect us. In the meantime, watch out for anyone on the train with a cold, you could be catching more than a stuffy nose!

The drug sibutramine, sold under the brand name Meridia, is known to increase blood pressure in a small number of individuals. This finding has made physicians reluctant to prescribe it to hypertensive patients, even if the high blood pressure is well controlled with medication. But recent study results indicate that those physicians can relax. A report published in the July 24 issue of the Archives of Internal Medicine found that controlled hypertensives who took sibutramine have no more likelihood of increased blood pressure than those who took placebo. The year long muticenter study randomized 224 controlled hypertensive subjects to receive either sibutramine or placebo for 52 weeks.

subjects. 224 healthy obese subjects (BMI 27-40) whose hypertension was controlled with a calcium channel blocker, and/or a diuretic. To qualify for the study, subjects needed to have their hypertension under control for at least 60 days prior to the initial screening visit. Patients were excluded for various medical conditions including cardiac disease, endocrine abnormalities, seizure, stroke, and drug allergies.

Method. After a 2- to 10-week run in period subjects were randomized to receive sibutramine (150) or placebo (74) for 52 weeks. The intial dosage in the sibutramine group was 5 mg once per day, titrated to 20 mg by week 6. The dose remained stable at 20 mg through week 52. Patients returned for a clinic visit every two weeks for the first 10 weeks of the study, and every 4 weeks thereafter. At clinic visits subjects were weighed and blood pressure was measured. Waist and hip measurements were made at baseline, week 28 and week 52. Triglycerides, HDL, LDL, total cholesterol, glucose and uric acid were measured at baseline and weeks 8, 28, and 32. Psychological tests were administered at baseline and weeks 8, 28, and 52.

Results.

Weight loss. Patients receiving sibutramine lost weight for the first six months of treatment, which was maintained until the end of the study. The mean change in body weight was -4.4 kg or 4.7 percent of body weight. Placebo patients lost an average of 0.5 kg. When divided by race, white patients lost 4.9 percent of body weight and black patients 4.0 percent. 40.1 percent of sibutramine subjects lost 5 percent or more of their body weight versus 8.7 percent for placebo; 13.4 percent of sibutramine subjects lost 10 percent or more of their body weight versus 4.3 percent for placebo.

Lipids, blood glucose and uric acid. Both 5 and 10 percent responders in the sibutramine group had significant improvements in triglycerides, HDL-C and glucose. Sibutramine also substantially decreased uric acid levels in all patients. High uric acid levels are associated with insulin resistance, high cholesterol and high triglycerides—risk factors for cardiovascular disease. (See table)

CHANGES IN SERUM LIPID, GLUCOSE AND URIC ACID
Variable	Sibutramine			Placebo
Variable	All patients	5% responders	10% responders	All patients	5% responders	10% responders
Triglycerides, mmol/L (no)	-0.19 (133)	-0.34 (57)	-0.49 (19)	-0.01 (59)	-0.14 (6)	-0.17 (3)
HDL-C, mmol/L (no)	0.14 (133)	0.24 (57)	0.28 (19)	0.06 (59)	0.32 (6)	0.47 (3)
LDL-C, mmol/L (no)	-0.09 (131)	-0.13 (55)	0.10 (17)	-0.11 (58)	-0.26 (6)	-0.25 (3)
Total cholesterol, mmol/L (no)	-0.03 (133)	0.03 (57)	0.23 (19)	-0.07 (59)	-0.01 (6)	0.14 (3)
Glucose, mmol/L (no)	0.23 (133)	0.61 (57)	-0.25 (19)	0.31 (59)	0.18 (6)	-0.26 (3)
Uric acid, µmol/L (no)	-17.84 (133)	-23.79 (57)	-47.58 (19)	-0.00 (59)	-17.84 (6)	-41.64 (3)

Blood pressure and pulse. Sibutramine patients had a small increase in diastolic blood pressure and pulse compared to the placebo group. But, significant increases in blood pressure were rare and comparable to patients receiving placebo. The mean body weight reduction, blood pressure and pulse were similar for the 81 black patients to those reported for white subjects. This is of particular interest to clinicians, as blacks are at increased risk of developing hypertension and obesity.

CHANGES IN BLOOD PRESSURE AND PULSE
Vital signs	Sibutramine			Placebo
Vital signs	All patients (n=142)	White patients (n=78)	Black patients (n=56)	All patients (n=69)	White patients (n=43)	Black patients (n=21)
Mean change in blood pressure, mm Hg
Systolic	2.7	3.9	0.1	1.5	0.7	2.5
Diastolic	2.0	2.4	1.2	-1.3	-0.3	-3.2
Mean change in pulse rate, /min at 3 consecutive visits, no. of patients (%)
>10-min Hg increase in systolic BP	24 (17)	17 (22)	5 (9)	9 (13)	4 (9)	4 (19)
>10-min Hg increase in diastolic BP	1 (1)	0 (0)	1 (2)	1 (1)	1 (2)	0 (0)
>10-min Hg increase in pulse rate	23 (16)	14 (18)	7 (12)	2 (3)	0 (0)	1 (5)

Cyberonics announced on August 7 that the first patient has been implanted with a bilateral NeuroCybernetic prosthesis, as part of a phase 1 obesity study. The pacemaker-like device curbs appetite by mildly stimulating the vagus nerve. The woman, who was implanted on Monday July 31, 2000 at Lenox Hill Hospital in New York, had no complications and was released from the hospital on August 5.

A total of six patents will be implanted in phase 1, with an additional 24 implantations scheduled for the second phase. The study will be conducted at up to four clinical sites including Lenox Hill Hospital and the University of Texas-Houston Medical School. The study will have an element of patient blinding. Some patients will receive stimulation shortly after implantation, while others will receive stimulation 3 months after the surgery.

For more on vagal nerve stimulation for obesity, see the July 2000 issue of Obesity-news.

Since our coverage of the phentermine shortage earlier this month, Obesity-news has uncovered two additional distributors of phentermine, both of which had a limited supply at the time we spoke with them. These companies have been added to the list of distributors in the August supplement. Although generally issues of on-line Obesity-news are not updated after publication, we will keep the manufacturer and distributor lists in the August supplement up-to-date so that subscribers will have a central location in which to look for sources.

PHENTERMINE DISTRIBUTORS
Company	Supply	Comments/Shipping policy
Martek Pharmacal Co. PO Box 264 Riverdale, NJ 07457 973/839-4840 Fax: 973/839-4744	yes	Has a limited supply.
Richie Pharmacal 1236 119 State Ave. PO Drawer 460 800/627-0250 Fax: 800/260-8153	yes	Has a limited supply of 15 mg capsules.

Obesity-news is following up with several leads on the phentermine shortage story, and will have further updates for you as new information becomes available. Updates will be published as a regular feature in the monthly newsletter until the shortage is over. In addition, if any vitally important information becomes available we will inform on-line subscribers by e-mail, and hard-copy only subscribers in a special mailing.

On August 7, the United Kingdom's Medicines Control Agency (MCA) reinstated licences for phentermine, which is sold under the brand names Duromine (phentermine hydrochloride) and Ionamin (phentermine resin) outside the US.

The reinstatement follows a challenge to the European Commission's decision that phentermine (Duromine, Ionamin) be withdrawn from the market. The European Court has suspended the Commission's decision until the Court makes a final ruling in the case. Following the Court's interim decision, the Medicines Control Agency has reinstated the licences of these products. The Committee on Safety of Medicines has advised that, as with amfepramone (diethylpropion), there are no major public health concerns in relation to phentermine products remaining on the UK market.

Mice expressing human uncoupling protein-3 (UCP-3) in skeletal muscle eat like hogs, but are thinner than control mice even though their activity levels are the same. This breakthough research is the first to show that UCP-3 may indeed be the gene that stimulates thermogenesis in humans. Investigators at SmithKline Beecham, the MRC Dunn Human Nutrition Unit and the University of Cambridge report that UCP-3 mice have only half as much fat even though they eat up to 50 percent more than normal mice. In addition, they have lower cholesterol, fasting glucose and insulin levels, and increased glucose clearance.

The quest for a gene that stimulates thermogenesis in skeletal muscle, the primary site of thermogenesis in humans, has been important in obesity drug discovery. In previous studies, it has come into question whether either UCP-2 or UCP-3 stimulate thermogenesis, since both are increased in starvation, a state associated with decreased energy expenditure.

The experiments.

In breeding the transgenic mice, several strains were generated. The UCP-3tg strain was selected for the experiments because expression of UCP-3 was increased 66 fold in skeletal muscle, but only minimally in brown fat.

UCP-3 mice are hyperphagic, eating 15 to 54 percent more than normal controls. UCP-3 mice fed a standard diet ate between 15 and 28 percent more than control mice from the ages of 4-8 weeks, 33-54 percent more on a high-fat diet from the ages of 8-12 weeks, and 50 percent more when returned to the standard diet. Even though they are hyperphagic, MRIs showed a significant decrease in fat mass of UCP-3tg mice after the high fat diet -- 44 percent in males and 57 percent in females. However, plasma leptin levels were not significantly reduced even with the severe reduction in fat. Despite the 50 percent increase in food consumption, plasma triglycerides and non-esterified fatty acids were similar in mutant and control mice, suggesting that fat combustion was higher in UCP-3tg mice.

Results consistent with an increase in energy expenditure. The decrease in fat in the UCP-3tg mouse is consistent with an increase in energy expenditure, as measured by resting oxygen consumption. After the normal diet oxygen consumption was 25 percent higher, and after the palatable diet it was 40 percent higher, without any increase in physical activity. Core body temperature was not affected, but muscle temperature was increased. The increased muscle temperature was similar to the increased BAT temperature following administration of b-3 agonists in rodents.

Thermogenesis a byproduct of fatty acid oxidation. While thermogenesis is increased in UCP-3tg mice, researchers suspect that it is a byproduct of greatly increased fatty acid oxidation. Other research has argued that UCP-1 mediated thermogenesis is also a byproduct of fatty acid oxidation, so that animals can survive on a low protein diet without becoming obese.

Conclusion.

Although some research fails to make an association between UCP-3 and thermogenesis, this study shows that overexpression of human UCP-3 produces a marked reduction in body weight and body fat, and improves insulin sensitivity. These results suggest that enhancement of UCP-3 expression, or stimulation of its activity, is a promising approach for the treatment of obesity.

Magainin Pharmaceuticals announced on July 28 that it has received a patent for an aminosterol, known as MSI-1436, for the treatment of obesity. Aminosterols are a class of shark molecule compounds originally discovered in the dogfish shark, Squalus acanthius.

Most aminosterols possess antibiotic activity, although the biologic functions in the shark are not well understood. Compound 7 (MSI-1436), has been studied extensively and induces profound appetite suppression and weight loss in mice, rats, dogs and monkeys without apparent toxicity. MSI-1436 may also be responsible for the sporadic feeding behavior of the dogfish which normally only eats every two weeks.

MSI-1436 produced weight loss in all rodent models of obesity.

In a rodent study, presented at the annual meeting of the Pediatric Academic Societies MSI-1436 caused significant appetite suppression resulting in the control of obesity and diabetes in leptin deficient (ob/ob), diabetic (db/db), MC-4 knockout, and agouti mice. The compound also reduced weight in wild-type mice and rats.

Rats given a single dose of MSI-1436 (10 mg/kg IV) reduced both food and fluid intake within 24 hours, and lost about 35 percent of their body weight by day 10 with no evidence of toxicity. Over a 4-month period MSI-1436 normalized insulin resistance, hypercholesterolemia and weight gain in ob/ob mice. Since all mutant mice responded similarly to MSI-1436, researchers suggest that its effects are independent of leptin and melanocortin.

MSI-1436, a novel drug.

MSI-1436 appears to act differently than any other appetite suppressant. The compound may interact with calmodulin, a calcium sensing protein, to alter calcium signaling within certain cells of the brain. Squalamine, now in Phase 2 cancer trials, is the first aminosterol discovered in the dogfish shark and works by sequestering calmodulin within the cell.

Alizyme plc announced on July 7 that it successfully completed a Phase 1a clinical trial of its obesity drug ATL-962. ATL-962 is a lipase inhibitor that works similarly to the drug Xenical.

In the trial 24 subjects received one of four dosage strengths of ATL-062 and 8 subjects received a placebo. All doses of ATL-962 were well tolerated, and no adverse events were observed. Alizyme will start phase 1b testing this fall, where repeat doses of ATL-962 will be administered.

ATL-962 is the only other lipase inhibitor being developed besides Hoffmann-LaRoche's Xenical. In pre-clinical studies the drug had similar efficacy to the Roche drug, and no toxicity was observed. Alizyme has established a "low cost production process" which may make the drug more affordable to consumers than Xenical. If the drug has fewer side effects in phase 1b, this could make it much more attractive as well.

Alizyme is developing ATL-962 in partnership with Oxford Molecular Group, a drug discovery outsourcing company.

Below is an update on the progress of drug research and new collaborations by major pharmaceutical companies since last October's, annual drugs in development issue.

Roche Pharmaceuticals announced that it is initiating a clinical study to evaluate the use of Xenical for overweight and obese teenagers. The multicenter trial will be conducted on approximately 450 obese subjects 12-16 years of age, who will receive either Xenical or placebo and a reduced calorie diet for one year. Approximately 25 percent of children in the United States are considered overweight, a figure that has doubled in the last 30 years.

Phytopharm and Pfizer have extended their collaborative research agreement for the development and commercialization of P57, an appetite suppressant derived for the extract of a South African plant.

Digital Gene Technologies (DGT) and the Joslin Diabetes Center at Harvard Medical School have announced that they will conduct joint research to identify genes responsible for the development of type-2 diabetes. The project will focus on the genes responsible for the onset and reversal of insulin resistance. Skeletal muscle appears to be the primary site of insulin resistance in type-2 diabetes, but the cellular mechanisms responsible remain unknown. The study will used DGT's patented TOGA gene expression profiling technology to evaluate changes in gene expression which occurring during treatment with rosglitazone, using type-2 diabetic subjects between the ages of 18 and 60 who require drug therapy due to uncontrolled diabetes.

Neurogen has entered into a licensing agreement Pfizer to develop drug candidates for the treatment of obesity in companion animals. The goal of the collaboration is to develop two types of small molecule drugs that will selectively block the NPY1 and NPY5 neurotransmitters. In a variety of animal studies, Neurogen has shown that selective NPY1 and NPY5 antagonists block NPY-induced feeding, producing significant weight loss.

Neurocrine Biosciences has received an NIH grant to support the development of melanocortin 4 receptor agonists. In phase 1 development, various new technologies will be used to optimize several compound families and develop lead molecules with higher potency. Neurocrine holds patents on MCRs 1-5 as part of its acquisition of Northwest Neurologic, founded by Dr. Roger Cone, one of the first researchers to clone the human form of the melanocortin receptors.

Gemini Genomics and Kyowa Hakko Kogyo Co. Ltd. have identified a gene responsible for obesity and type 2 diabetes. The gene which is thought to be involved in the development of several age-related diseases, like osteoporosis and emphysema, was also found to be associated with insulin resistance syndrome, a cluster of conditions which lead to the development of type-2 diabetes and cardiovascular disease.

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IMPORTANT: All information in this publication is believed to be accurate and true. Publisher is not liable for omissions or inaccuracies. Information in this newsletter is for educational purposes only and should not be construed as medical advice, or be used in lieu of consultation with a health care provider.

LETTER FROM THE EDITOR.

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