LG839: Anti-obesity effects and polymorphic gene correlates of reward deficiency syndrome

Abstract

IntroductionThis study systematically assessed the weight management effects of a novel experimental DNA-customized nutraceutical, LG839
(LifeGen®, Inc., La Jolla, CA, USA).

MethodsA total of 1058 subjects who participated in the overall D.I.E.T. study were genotyped and administered an LG839 variant based
on polymorphic outcomes. A subset of 27 self-identified obese subjects of Dutch descent, having the same DNA pattern of four
out of the five candidate genes tested (chi-square analysis) as the entire data set, was subsequently evaluated. Simple t tests comparing a number of weight management parameters before and after 80 days of treatment with LG839 were performed.

ResultsSignificant results were observed for weight loss, sugar craving reduction, appetite suppression, snack reduction, reduction
of late night eating (all P<0.01), increased perception of overeating, enhanced quality of sleep, increased happiness (all P<0.05), and increased energy (P<0.001). Polymorphic correlates were obtained for a number of genes (LEP, PPAR-γ2, MTHFR, 5-HT2A, and DRD2 genes) with positive clinical parameters tested in this study. Of all the outcomes and gene polymorphisms, only the DRD2
gene polymorphism (A1 allele) had a significant Pearson correlation with days on treatment (r=0.42, P=0.045).

ConclusionIf these results are confirmed in additional rigorous, controlled studies, we carefully suggest that DNA-directed targeting
of certain regulator genes, along with customized nutraceutical intervention, provides a unique framework and strategic modality
to combat obesity.

Full text

Page 1

Adv Ther. 2008;25(9):894-913.
DOI:10.1007/s12325-008-0093-z
LG839: Anti-Obesity Effects and
Polymorphic Gene Correlates of
Reward Deficiency Syndrome
894

ORIGINAL RESEARCH
Kenneth Blum
Department of Physiology & Pharmacology, Wake Forest University
School of Medicine, Winston-Salem, North Carolina, USA
LifeGen®, Inc., La Jolla, California, USA
PATH Medical & Research Foundation, New York, New York, USA
Synaptamine™, Inc., San Antonio, Texas, USA
Amanda L. C. Chen
Thomas J. H. Chen
Chang Jung Christian University, Tainan, Taiwan
Changhua Christian Hospital, Changhua, Taiwan
Patrick Rhoades
Comprehensive Obesity Clinic, Modesto, California, USA
Thomas J. Prihoda
Department of Pathology, University of Texas Health Science Center,
San Antonio, Texas, USA
B. William Downs
Roger L. Waite
Lonna Williams
LifeGen®, Inc., La Jolla, California, USA
Address correspondence to: Dr. Kenneth Blum, Department of Physiology &
Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd.,
Winston-Salem, North Carolina, USA. Email: drd2gene@aol.com
Address correspondence to: Amanda L. C. Chen, Chang Jing Christian University,
Tainan, Taiwan. Email: tjhchen@yahoo.co.tw

Page 2

Blum et al.
895
ABSTRACT
Introduction: This study systematically assessed the weight management
effects of a novel experimental DNA-customized nutraceutical, LG839
(LifeGen®, Inc., La Jolla, CA, USA).
Methods: A total of 1058 subjects who participated in the overall D.I.E.T.
study were genotyped and administered an LG839 variant based on poly-
morphic outcomes. A subset of 27 self-identified obese subjects of Dutch
descent, having the same DNA pattern of four out of the five candidate
genes tested (chi-square analysis) as the entire data set, was subsequent-
ly evaluated. Simple t tests comparing a number of weight management
parameters before and after 80 days of treatment with LG839 were per-
formed.
Results: Significant results were observed for weight loss, sugar crav-
ing reduction, appetite suppression, snack reduction, reduction of late
night eating (all P<0.01), increased perception of overeating, enhanced
quality of sleep, increased happiness (all P<0.05), and increased energy
Eric R. Braverman
Department of Neurosurgery, Weill Cornell Medical College,
New York, New York, USA
PATH Medical & Research Foundation, New York, New York, USA
Dasha Braverman
Vanessa Arcuri
Mallory Kerner
PATH Medical & Research Foundation, New York, New York, USA
Seth H. Blum
Synaptamine™, Inc., San Antonio, Texas, USA
Tomas Palomo
Hospital Universitario, 12 de Octubre, Servicio de Psiquiatria Av.
Cordoba SN, Madrid, Spain

Page 3

Advances in Therapy®
Anti-Obesity Effects of LG839
896
INTRODUCTION
Obesity was formerly characterized as
purely a behavioral disorder involving poor
dietary habits and lack of physical exercise,
due in large part to a lack of willpower
or self-restraint.1 Over the past decade,
however, the consensus of the medical
community has evolved and the Institute
of Medicine at the National Academy of
Sciences, the National Institute of Health
(NIH), and the International Classifica-
tion of Diseases, among many others, now
recognize obesity as a chronic, multifacto-
rial medical disease of energy metabolism
and appetite regulation involving genetics,
environment, physiology, and biochemis-
try.1-3 However, in persons not classified
as obese but rather as overweight, the US
Food and Drug Administration (FDA) re-
cently noted in its final rules under the Di-
etary Supplements Health and Education
Act of 1994, that despite obesity being a
disease, a person being overweight but less
than obese refers not to a disease but to the
structure and function of the body.4
Obesity-related medical conditions
contribute to 300,000 deaths in the United
States each year, second only to smoking as
a cause of preventable death; and obesity
has been established as a major risk factor
for hypertension, cardiovascular disease,
type 2 diabetes, and some cancers in both
men and women.5
Obesity affects an estimated 58 mil-
lion people in the United States and its
prevalence is increasing.6 Approximately
one-third of American adults are estimat-
ed to be obese, and 60% are overweight.7
In response to this rising epidemic, the
medical, food, and fitness communities
have consistently advised the population
to make behavioral modifications, such as
improving diet and exercising. However,
as people have changed their eating and
exercise habits, the rates of obesity have
continued to rise. The combined preva-
lence of overweight and obese persons
in the United States has increased from
46% of the adult populations (National
Health and Nutrition Examination Sur-
vey [NHANES] II, 1976-1980) to over
(P<0.001). Polymorphic correlates were obtained for a number of genes
(LEP, PPAR-γ2, MTHFR, 5-HT2A, and DRD2 genes) with positive clini-
cal parameters tested in this study. Of all the outcomes and gene polymor-
phisms, only the DRD2 gene polymorphism (A1 allele) had a significant
Pearson correlation with days on treatment (r=0.42, P=0.045).
Conclusion: If these results are confirmed in additional rigorous, con-
trolled studies, we carefully suggest that DNA-directed targeting of cer-
tain regulator genes, along with customized nutraceutical intervention,
provides a unique framework and strategic modality to combat obesity.
Keywords: LG839; neurotransmitters; obesity; reward deficiency
syndrome; sugar craving behavior; weight loss

Page 4

Blum et al.
897
60% of the adult population in NHANES
III (1988-1994).7 A 2006 survey suggest-
ed that people who are obese have similar
eating and exercise habits, as well as similar
behaviors in snacking, reading nutritional
labels, and eating out, when compared
with normal weight people.7
Despite NIH, World Health Organi-
zation, and National Academy of Sciences
statements that genetic factors play a role
in obesity, a genetic test has not yet been
developed to diagnose or stratify this con-
dition, nor have there been treatments to
target genetic associations. To address this
unmet clinical need, our laboratory has
developed a patent-protected genetic test
and companion customized nutritional
product called LG839 (LifeGen®, Inc., La
Jolla, CA, USA).8 The basic components
consist of Dl-phenylalanine, L-tyrosine,
5-hydroxytryptophane, L-glutamine, chro-
mium salts, vitamin B6, rhodiola rosea,
and passion flower.
Our previous research into alcoholism,
another prevalent healthcare condition
that has also been traditionally character-
ized in behavioral terms, suggested that
the condition had a genetic component.9
This research, and work by other labora-
tories, continued to explain how analyz-
ing certain genotypes could guide dietary
intake and environments to overcome
this genetic predisposition.9 Similar to al-
coholism, obesity may be due in part to
certain genetic predispositions, and by the
body engaging in behavior to overcome
these deficiencies, the host may engage in
behaviors that are individually unhealthy
or detrimental. In fact, it has been shown
that the prevalence of polymorphisms of
the DRD2 receptor gene significantly in-
creases in obese subjects with comorbid
substance use disorder.10
This initial research began to elucidate
a concept that has been defined as reward
deficiency syndrome (RDS). RDS results
from a dysfunction in the brain reward
cascade (Figure 1), which directly links
abnormal craving behavior with a defect
in the DRD2 dopamine receptor gene
as well as in other dopaminergic genes
(D1, D3, D4, D5).12-14 A number of other
candidate genes are also associated with re-
ward dependence in the neurogenetics of
RDS.15 However, dopamine is a very pow-
erful neurotransmitter in the brain that
controls feelings of well-being produced
through the interaction of dopamine and
neurotransmitters such as serotonin and
the opioids.15 The complex interactions of
these neurotransmitters ultimately regu-
late the dopaminergic activity in the me-
solimbic system of the brain, which has
been termed the brain reward cascade.11
RDS involves a form of sensory de-
privation of the brain’s reward or pleasure
mechanisms and is manifested as a con-
sequence of an individual’s biochemical
inability to derive reward from ordinary,
everyday activities. The concept of RDS
can explain how simple genetic anomalies
give rise to complex aberrant behavior.16,17
In this regard, other genetic polymor-
phisms have been associated with obe-
sity, including the dopamine D2 receptor
(DRD2), methylenetetrahydrofolate re-
ductase (MTHFR), leptin (LEP), seroton-
ergic receptor (5-HT2A), and peroxisome
proliferator-activated receptor gamma 2
(PPAR-γ2) genes.18-23

Page 5

Advances in Therapy®
Anti-Obesity Effects of LG839
898
Serotonin (5-HT)
Dopamine (DA)
Dopamine (DA)
GABA
GABA A receptor
Norepinephrine
Receptor
D2 receptor
Dopamine (DA)
D2 receptor
Dopamine (DA)
REWARD
REWARD
REWARD
GABA
Receptor
Figure 1. Brain reward cascade. In this cascade stimulation of the serotonergic system in the hypothala-
mus leads to the stimulation of delta/mu receptors by serotonin to cause a release of enkephalins.
Activation of the enkephalinergic system induces an inhibition of GABA transmission at the substantia
nigra by enkephalin stimulation of mu receptors at GABA neurons. Tis inhibitory efect allows for the
fne-tuning of GABA activity. Tis provides the normal release of dopamine at the projected area of the
nucleus accumbens (reward site of the brain).11
Figure reproduced with permission fom Blum K, Kozlowski GP. Ethanol and neuromodulator interactions:
a cascade model of reward. In: Ollat H, Parvez S, Parvez H, eds. Alcohol and Behavior. Utrecht: VSP Press;
1990:131-149.
L. coeruleus (A6)
Hippocampus
Substantia nigra
Amygdala
Nucleus
accumbens
Enkephalins
Receptor
Enkephalin
Opiate receptor
GABA
GABA B receptor
Subtantia
nigra A9
Nucleus
accumbens
Locus
coeruleus
A6
Hypothalamus
HypothalamusVentral
tegmental
region (A10)
inhibits
inhibits
releases
releases
Amygdala
Hippocampus
CAI cluster
cells
CAx cluster
cells
Ventral tegmental
region (A10)
Neuropeptidases
Norepinephrine

Page 6

Blum et al.
899
Preliminary,
studies with an LG839 variant called
PCAL-103 in obese and overweight sub-
jects indicated that PCAL-103 facilitated
withdrawal from carbohydrate bingeing,
reduced relapse rates, and increased weight
loss compared with controls.24 Subsequent
prospective studies have demonstrated
that PCAL-103 significantly increased
weight loss, decreased food cravings and
binge eating, prevented weight regain after
a period of fasting, and also reduced stress
and enhanced general well-being.25,26
In light of these early hypothesis-gen-
erating studies, and the paucity of research
involving neurotransmitter manipulation
of the brain reward system coupled with
genetic polymorphic identification, we de-
signed a pilot study to evaluate the process
of DNA-customization of a nutritional so-
lution for weight management. We carried
out this experiment cognizant that larger
randomized, placebo-controlled studies
are warranted before any solid conclusions
can be drawn from the present evaluation.
hypothesis-generating

MATERIALS AND METHODS
Subjects
The first stage of the study involved a
broad sample of 1058 subjects who par-
ticipated in the overall D.I.E.T. study
and who had self-identified themselves
as obese or overweight. All subjects were
recruited from customers who had pur-
chased LG839 as part of a commercial pi-
lot in the Netherlands from January 2006
to February 2007, and study participants
were approached without a particular
schema. The study subjects were primarily
an ethnically homogenous group of Dutch
descent. The second stage of the study in-
volved a narrow sample of 27 subjects who
had self-identified themselves as obese or
overweight. These subjects participated in
the LG839 pilot in the Netherlands, were
willing to complete a retrospective survey
about their results, and were willing to par-
ticipate without compensation.
Study Design
This cross-sectional, observational
study included a genetic analysis involving a
panel of genes, and an evaluation question-
naire providing two key self-reported ret-
rospective data sets on the subjects’ health
before taking LG839 and then after taking
LG839. Self-reported evaluations were col-
lected through an online questionnaire dis-
seminated over email to study participants.
Study Outcomes
The primary objective was to evaluate
the safety and efficacy of LG839 in obese
and overweight subjects, and specifically to
look at differences in weight, mood, sleep,
digestion, and other health issues before
and after taking LG839. All participants
provided written consent, and the study
protocol was approved by the institutional
review board of the nonprofit research or-
ganization, PATH Medical & Research
Foundation, located in New York, USA.
Data were collected online independently
by LooseFoot Computing Ltd. of Regina,
Saskatchewan, Canada.

Page 7

Advances in Therapy®
Anti-Obesity Effects of LG839
900
Laboratory Measurements and
Genotyping
Laboratory testing was performed in
a high-complexity, CLIA-certified labo-
ratory under contract with the company
based in San Diego, California, USA. All
subjects were genotyped based on a neutral
identification number and read without
knowledge of the individual being typed.
Total genomic DNA was extracted from
each coded blood sample, and aliquots
were used for polymerase chain reaction
(PCR) analysis.
DRD2
The DRA1 and DRA2 genotyping
was performed using PCR.9,27 The oligo-
nucleotide primers 5΄-CCGTCGAC-
CCTTCCTGAGTGTCATCA-3΄ and
5΄CCGTCGACGGCTGGCCAAGT-
TGTCTA-3΄ were used to amplify a
310-base pair (bp) fragment spanning the
polymorphic Taq1A1 site of the DRD2
gene. PCR was performed in 30 mL reac-
tion mixtures containing 1.5 mM MgCl2,
2 mM 2΄-deoxynucleotide 5΄-triphosphates,
0.5 µM primers, 1 µg of template DNA,
1.5 U Taq polymerase (Boehringer Man-
nheim Corp., IN, USA), and PCR buffer
(20 mM Tris-HCL [pH 8.4] and 50 mM
KCl). After an initial denaturation at 94°C
for 4 min, DNA was amplified with 35 cy-
cles of 30 sec at 94°C, 30 sec at 58°C, and
30 sec at 72°C, followed by a final extension
step of 5 min at 72°C. The PCR product
was digested with 5 U of Taq1 for 22 hours
at 65°C for the Taq1A polymorphism.
Digestion products were then resolved
on a 3% agarose gel (5 V/cm) containing
0.65 µg/mL ethidium bromide. There were
three DRD2 Taq1A genotypes: 1) the pre-
dominant homozygote A2/A2, which ex-
hibits two restriction fragments of 180 and
130 bp; 2) the heterozygote A1/A2, which
exhibits three restriction fragments of 310,
180, and 130 bp; and 3) the rare homozy-
gote A1/A1, which produces only the un-
cleaved 310-bp fragment.
MTHFR
Genotyping for the MTHFR C677T
polymorphism was performed using
PCR and restriction fragment length
polymorphism analysis. We designed
PCR primers 5΄-CCCAGCCACT-
CACTGTTTTAG-3΄
GAAGAACTCAGCGAACT-3΄
DNASIS Pro Ver.2.0 (Hitachi Software
Engineering Co. Ltd., Tokyo, Japan).
Since the C to T transition at nucleotide
677 produces a Hinf I digestion site, the
amplified 469-bp product derived from
the mutant gene was cleaved into 393-bp
and 76-bp fragments by Hinf I (TaKaRa
Bio Inc., Shiga, Japan), which leaves the
wild type gene unaffected. After electro-
phoresis through a 6% polyacrylamide gel,
the digestion products were visualized by
staining with ethidium bromide.
and 5΄-TGG-
with
5-HT2A
Genotyping of the –1438G/A poly-
morphism of the 5-HT2A gene was carried
out by PCR and restriction digestion as
described previously.20 Genomic leukocyte
DNA (100 ng in a final volume of 10 μL)

Page 8

Blum et al.
901
was amplified by PCR using the following
primers: 5΄-AAGCTGCAAGGTAG-
CAACAGC-3΄ and 5΄-AACCAACT-
TATTTCCTACCAC-3΄. The primers
amplified a product of 468 bp. The PCR
conditions were as follows: an initial dena-
turation step at 94°C for 3 min, followed
by 35 cycles of denaturation at 94°C for
15 sec, annealing at 55°C for 15 sec, and
extension at 72°C for 30 sec, with a final
extension of 10 min at 72°C. The PCR
reaction product was digested at 37°C
overnight with 5 U of the restriction en-
zyme MspI (New England Biolabs, MA,
USA). The –1438G allele was cut into
244-bp and 224-bp fragments, whereas
the –1438A allele remained undigested.
The fragments were separated on a 2%
agarose gel and visualized by staining with
ethidium bromide.
PPAR-γ2
Two hundred base pairs of sequence
surrounding PPAR-γ2 Pro12Ala were pro-
vided to Applied Biosystems (Foster City,
CA, USA) to develop TaqMan allelic
discrimination assays using their assay by
design platform. Genotyping of the Pro-
12Ala AD was performed using the fol-
lowing primers (0.9 mol/L each): forward
5΄-TTATGGGTGAAACTCTGGGA-
GATT-3΄ and reverse 5΄-TGCAGA-
CAGTGTATCAGTGAAGGA-3΄ and
the Taqman MGB probes Fam-TTCT-
GGGTCAATAGG and Vic-CTTTCT-
GCGTCAATAG (0.1 mol/L each;
Applied Biosystems). Four nanoliters of
a 10 ng/L stock of DNA was dispensed
into 384-well PCR plates using a Biomek
FX robot (Beckman Coulter, Fullerton,
CA, USA), to which 6 µL of a mix con-
taining primers, TaqMan MGB probes,
and TaqMan Universal PCR Master Mix
(Applied Biosystems) were added. These
were sealed with optical seals (Applied
Biosystems) and incubated at 95°C for
10 min, followed by 40 cycles of 95°C for
15 sec, and 60°C for 1 min before analy-
sis on a 7900HT plate reader (Applied
Biosystems). Individual genotypes were
determined using SDSv2.1 software (Ap-
plied Biosystems).
LEP
We used the following dinucleotide re-
peats (D7S1873, D7S1875, D7S514, and
D7S680) present on the YAC contig con-
taining the human LEP gene as described
by Green.28 The primers used for PCR
were the same as in their report. Of these,
D7S1875 was closest to the LEP gene.
We refer to this as LEP1875. The frequency
of the different alleles of the LEP1875 poly-
morphism has been reported previously.27
The alleles ranged in size from 199 to
225 bp in length. Our a priori approach to
the examination of dinucleotide repeats
in behavioral disorders is based on other
research.29,30
Comings et al.27 was first to divide the
alleles into approximately equal groups,
since there was a natural tendency for
a bimodal distribution, which was the
case for the LEP1875 polymorphism and
the cut was made at 208 bp. The result-
ing genotypes were <208 bp/<208 bp,
<208 bp/≥208 bp, and ≥208 bp/
≥208 bp. In this experiment we used

Page 9

Advances in Therapy®
Anti-Obesity Effects of LG839
902
Coming’s cut-off point in our analysis
providing for only positive and negative
allelic associations.
Nutraceutical Customization
Based upon the genetic profiles de-
rived, certain ingredients in LG839 were
customized according to subjects’ geno-
types (Table 1).
Measurements of Efficacy and Safety
Separate from the laboratory measure-
ments, all measurements of efficacy and
safety were self-reported by the study sub-
jects using a one-time retrospective online
questionnaire. The primary endpoint was
weight loss in kilograms. Secondary end-
points included changes in appetite, sugar
cravings, waist circumference, mood, sleep,
and digestion. The changes in these factors
were measured generally using a scale of 1 to
5 where 5 was the least healthy rating and
1 was the most healthy rating to provide
consistency across the multiple measures.
Tertiary endpoints included changes in in-
cidences of depression and cravings for alco-
hol/tobacco/recreational drugs. To monitor
persistency, we asked study subjects to self-
report on their frequency of compliance to
the product’s serving instructions.
Table 1. Customization of the nutraceutical LG839 according to genotype.
Genotype
MTHFR (C677T)
Homozygous or normal/wild type (CC)31,32
MTHFR (C677T)
Heterozygous mutant (CT)
or homozygous mutant (TT)33
5-HT2A (1438A)
Heterozygous or normal34
5-HT2A (1438A)
Heterozygous or homozygous
mutant or leptin LEP1875
DRD2
Wild type A2/A235-37

DRD2
Heterozygous A1/A2
or homozygous A1/A138
5-HT2A=serotonergic receptor; DRD2=dopamine D2 receptor; LEP=leptin;
MTHFR=methylenetetrahydrofolate reductase.
Ingredient
Vitamin B9 (folic acid)
Serving
800 μg
Vitamin B9 (folic acid) 5 mg
SuperCitrimax (–)hydroxycitric acid
(extract of Garcinia cambogia)
SuperCitrimax (–)hydroxycitric acid
(extract of Garcinia cambogia)
2500 mg
4667 mg
35
Synaptamine™ complex (DL-phenylalanine,
chromium, L-glutamine, L-tyrosine, L-5-HTP,
vitamin B6 [pyridoxal-5-phosphate])
Synaptamine™ complex (DL-phenylalanine,
chromium, L-glutamine, L-tyrosine, L-5-HTP,
vitamin B6 [pyridoxal-5-phosphate])
1725 mg
2750 mg

Page 10

Blum et al.
903
Statistical Analysis
The means and standard deviations
were calculated before and during LG839
treatment for each item in the question-
naire. To test for a change, the paired t test
was used on the change score, with change
calculated as response during treatment
minus the response before treatment for
each item. Significance was denoted at
each level of 0.05, 0.01, and 0.001 in the
results.
Two-sample t tests were used with
Satterthwaite’s approximate t test when
the F test for equal variances rejected
for comparing changes in each item for
those with each genetic polymorphism
compared to those without the polymor-
phism.
Genotypic and allelic distributions
were compared with the Pearson chi-
square test for homogeneous variances.
Three groups were compared: historical
literature controls, the sample of 1058,
and the subset for this analysis (n=27).
The effect of each polymorphism on quan-
titative variables was tested using multiple
linear regression. Pearson’s correlation
was used to determine which responses
and which genetic polymorphisms were
related to days on treatment. Stepwise
multiple regression was used to determine
the model for each outcome considering
the five genetic polymorphisms, days on
LG839 treatment, and extent to which
subjects followed instructions. R2 cor-
relations were given for the final model’s
ability to explain variance in the outcome
measurement. These were contribution to
the overall variance of a preassigned total
sum outcome index (weight loss in kilo-
grams, reduction in waist circumference
in inches, sugar cravings, snack intake, late
night bingeing behavior, exercise, appe-
tite, and energy level). In addition, Cron-
bach’s alpha was used to determine that
the index sum indeed was reliable. It was
reliable, as Cronbach’s alpha was 0.73 for
the total sum of change scores. This total
provides an overall response to use for the
multiple regression, in addition to the in-
dividual item outcomes measured in the
questionnaire.
Percentages of each gene polymor-
phism for the self-identified sample of
obese (n=1058) and for the variable sam-
ple size of normal controls from the liter-
ature were each compared to the sample
(n=27) in this study for each gene poly-
morphism individually by Fisher’s exact
test.39 We also pooled the genotypes from
each group (1058 obese subjects; 27 obese
subjects [subset]; literature controls) and
statistically compared genotypic patterns
for significant differences.
The Statistical Analysis System (SAS
Institute, 2006, version 9.1 for the PC,
Cary, North Carolina, USA) was used for
these calculations.
RESULTS
Significant improvements were ob-
served for weight loss, sugar craving
reduction, appetite suppression, snack re-
duction, reduction of late night eating (all
P<0.01), increased perception of overeat-
ing, enhanced quality of sleep, increased
happiness (all P<0.05), and increased en-
ergy (P<0.001) (Table 2).

Page 11

Advances in Therapy®
Anti-Obesity Effects of LG839
904
It is to be noted that because of miss-
ing values for the genotyping statistics
(n=23), even with this small sample size,
we obtained significant polymorphic cor-
relates with regard to a number of well-
known genes (LEP, PPAR-γ2, MTHFR,
5-HT2A, and DRD2). In this regard,
carrying certain known polymorphisms
(MTHFR [C677T allele]; LEP [D7S1875
allele]; 5-HT2A [1438G/A allele]; DRD2
[A1 allele]; PPAR-γ2 [Pro12Ala allele])
correlated negatively with positive clinical
parameters tested in this study. The per-
centage prevalence for each gene polymor-
phism is presented in Table 3.
A chi-square test was used to deter-
mine whether a genetic difference occurred
between the 1058 subjects participating
in the overall D.I.E.T. study, the histori-
cal literature controls, and the subset of
participants (n=27) utilized in the pres-
ent study. The test revealed no difference
Table 2. Overall changes in obesity-associated health measures.
Assessment area and question
n
25
Mean change* SD
P
Anxiety –0.32 0.85 0.073
Appetite 25 –0.76 1.20 0.004
Drug craving 25 –0.20 0.71 0.170
Tobacco craving 25 –0.00 0.00 NA
Depression 25 –0.20 0.58 0.096
Ease of sleep 25 –0.28 1.02 0.183
Energy 25 –0.84 1.03 0.0004
Exercise 25 +0.56 2.02 0.179
Get along with others 25 +0.08 0.49 0.425
Happiness 25 –0.36 0.70 0.017
Interrupted sleep 25 –0.48 1.61 0.149
Late night eating 25 –0.84 1.43 0.007
Perception of overeating 25 –0.72 1.49 0.023
Quality of sleep 25 –0.52 1.00 0.016
Regularity of digestion 25 –0.40 1.04 0.067
Snacking 25 –0.80 1.29 0.005
Sugar craving 25 –0.88 1.51 0.008
Waist, cm 16 –0.75 1.73 0.104
Weight, kg 26 –2.62 4.55 0.006
*Responses during LG839 administration minus baseline responses.
NA=not applicable.

Page 12

Blum et al.
905
between any gene polymorphism evalu-
ated for those present in each individual
group compared to the average for all three
groups. Some gene polymorphisms differed
among the three groups, but the subset of
n=23 (reflecting missing data) always had
a percentage near the average for all three
groups and thus did not differ from the
overall gene polymorphism distribution.
These percentages were 31.3% overall for
the DRD2 gene and 39.1% for the subset.
The 5-HT2A receptor gene was present in
68.4% overall and 64.0% in the subset. The
PPAR-γ2 gene was present in 17.5% over-
all and 17.4% for the subset. The MTHFR
gene was present in 69.9% overall and 64.0%
for the subset. The LEP gene was present in
70.4% overall, but in only 56.0% of the sub-
set. This was the largest difference, but the
subset is intermediate to the high sample of
1058 (75.6%) and low sample of literature
controls (n=206, 45.6%) and its deviation
of 56.0% positive compared to the aver-
age of 70.4% positive does not contribute
significantly to the overall chi-square test.
However, when we compared each group
separately against each other and controls
we found similar results, except for the LEP
gene (Table 3).
The significant results are as follows:
PPAR-γ2 was significantly associated with
increased interruptions of sleep (P<0.04)
as well as reduced hours of exercise
(P<0.02); MTHFR was significantly asso-
ciated with weight loss resistance (P<0.05);
5-HT2A was significantly associated with
loss of waist circumference resistance
(P<0.008); and DRD2 was significantly
associated with reduced hours of exercise
(P<0.04). We found no association with
the LEP gene polymorphism for any
parameter tested.
Table 3. Genotypic comparisons between the 1058 obesity sample, the subset of 27 obese subjects,
and historical literature controls*.
Name and Prevalence (%), mutant (combined heterozygous/homozygous) Frequency Frequency
description of Subset of self-selected Obesity Literature for wild for mutant
gene studied obese subjects sample control sample type type
PPAR-γ2
5-HT2A
MTHFR
DRD2
LEP‡
*Due to missing data the mean was n=23.
†Signifcantly diferent from n=23 sample by Fisher’s exact test.
‡We are cognizant that many studies on the LEP gene argued its association with obesity.
5-HT2A=serotonergic receptor; DRD2=dopamine D2 receptor; LEP=leptin;
MTHFR=methylenetetrahydrofolate reductase; PPAR-γ2=peroxisome proliferator-activated
receptor gamma 2.
4/23 (17.4) 265/1058 (25.0) 314/2244 (14.0) 0.90 0.10
16/25 (64.0) 679/1058 (64.2) 173/283 (61.1) 0.56 0.44
16/25 (64.0) 739/1058 (69.9) 54/100 (54.0) 0.60 0.40
9/23 (39.1) 403/1058 (38.1) 945/3258 (29.0) 0.76 0.24
14/25 (56.0) 800/1058 (75.6)† 94/206 (45.6) 0.60 0.40

Page 13

Advances in Therapy®
Anti-Obesity Effects of LG839
906
The multiple regression analysis iden-
tified several outcomes that could be
jointly predicted by multiple variables.
Both days on treatment and compliance
with instructions jointly predicted exer-
cise change (r2=0.58, P=0.0004). Both
the PPAR-γ2 gene polymorphism and
compliance with instructions jointly pre-
dicted change in getting along with others
(r2=0.42, P=0.007). Both the 5-HT2A
gene and LEP gene jointly predicted
waist size change (r2=0.56, P=0.024).
All other outcomes related to this study
were only significant for the univariate
results given earlier. Of all the outcomes
and gene polymorphisms, only the DRD2
gene polymorphism had a significant Pear-
son correlation with days on treatment
(r=0.42, P=0.045).

DISCUSSION
In the present study, albeit with a small
sample size, we showed significant effects on
multiple obesity-related parameters using
the experimental DNA-customized nutra-
ceutical LG839. The most robust findings
were for weight loss, sugar craving reduc-
tion, appetite suppression, snack reduction,
and reduction of late night bingeing. Other
significant findings included increased per-
ception of overeating, increased energy,
increased quality of sleep, and overall hap-
piness. Of even greater significance was the
finding that certain gene polymorphisms
correlated with resistance to a number of
outcome measures including weight loss,
loss of waist circumference, exercise, and
sleep interruptions. It appears that in the
presence of these polymorphic correlations,
LG839 induced significant positive changes
suggesting anti-obesity activity.
These results are consistent with earlier
reports involving LG839 variants.24-26 Fur-
thermore, a loss of 2.5 kg in only 80 days
is quite statistically robust especially when
considering guidelines from the US FDA
regarding meaningful weight loss of any
medically approved anti-obesity pharma-
ceutical.
The NIH 1998 Clinical Guidelines on
the Identification, Evaluation, and Treat-
ment of Overweight and Obesity in Adults
defines normal weight as a body mass index
(BMI) of 18.5-24.9 kg/m2, overweight as a
BMI of 25-29.9 kg/m2, and obese as a BMI
of ≥30 kg/m2. The Clinical Guidelines rec-
ommend weight loss through a combina-
tion of diet modification, increased physical
activity, and behavior therapy for obese pa-
tients, and for patients who are overweight
or have a high-risk waist circumference,
when accompanied by two or more risk fac-
tors. In the event that lifestyle changes do
not promote weight loss after 6 months, the
Clinical Guidelines recommend that drugs
should be considered as adjunctive therapy
for select patients who have a BMI of ≥30
kg/m2, or a BMI of ≥27 kg/m2 if concomi-
tant obesity-related risk factors or disease
exist. The FDA’s approach to the approval
of prescription weight-loss drugs mirrors
the recommendations provided in the
NIH’s Clinical Guidelines. For example, the
prescription of orlistat was studied and ap-
proved for long-term weight loss in patients
moderately-to-severely overweight (BMI
27-29.9 kg/m2) with comorbid conditions
such as hypertension, type 2 diabetes, or
dyslipidemia, and in obese subjects (BMI

Page 14

Blum et al.
907
≥30 kg/m2) regardless of the presence of
comorbidities. Limiting approval of pre-
scription weight-loss drugs to moderately-
to-severely overweight individuals with
comorbidities and to obese patients maxi-
mizes the therapeutic risk-benefit profile by
targeting drug therapy to individuals whose
risk for weight-related disease is high and
likely to outweigh the risks associated with
any given pharmacological agent. In clinical
studies, orlistat demonstrated weight loss of
5% or more at 6 and 12 months.40 In light of
the current results, which suggest a potential
weight loss of 2.5% in 80 days, or less than
3 months, the LG839 product warrants
further investigation to evaluate whether
these pilot study results are replicable in a
randomized, placebo-controlled study of a
longer duration.
Gene Polymorphic Correlates
Obesity is a biogenetic condition in-
volving both environmental and genetic
antecedents. Similar to diseases such as al-
coholism that were once classified as pure-
ly behavioral abnormalities, obesity is now
accurately characterized as a disease with
biological and genetic factors. It has been
reported that in both Caucasian women
and men, heritability for BMI was 0.58
and 0.63; for body fat percentage, 0.59 and
0.63; and for lean body mass 0.61 and 0.56,
respectively.41 Because of the complexity
of obesity and related conditions involving
reward deficiencies, it is likely that more
than one defective gene is involved. How-
ever, work from Volkow’s laboratory using
PET scanning showed a DRD2 paucity in
subjects with RDS versus non-RDS sub-
jects.42 It is of note that in this pilot study
we found significant results for a number
of gene-outcome parameters.
PPAR
In terms of gene associations, it is
noteworthy that the development and
metabolism of adipocytes are regulated in
a complex manner and the important role
of the transcription factor PPAR-γ has
been recently recognized.22,23 PPAR-γ is a
member of the nuclear hormone receptor
family of transcription factors, which are
known to be involved in transcription of
target genes.22,23 PPAR-γ2 is specific for
adipose tissue; this characteristic makes it
a candidate gene for the regulation of in-
sulin and glucose metabolisms.43 Among
genetic variants identified in the PPAR-γ2
gene, the Pro12Ala variant is highly
prevalent in Caucasians.43 Conflicting
results regarding the association between
this mutation and complex traits, such as
obesity, insulin sensitivity, and type 2 dia-
betes have been reported. Some reports
suggest that the X12Ala (either Pro12Ala
or Ala12Ala) genotype is related to im-
proved insulin sensitivity, particularly
in subgroups with obesity, although this
conclusion is still controversial.22-24,44-47
Interestingly, we found a negative associa-
tion between the Pro12Ala or Ala12Ala
polymorphism of the PPAR-γ2 gene and
both quality of sleep and exercise. In fact,
there are a number of studies relating the
PPAR-γ2 gene and exercise.43,48 However,
this is the first report linking sleep inter-
ruption and the Pro12Ala polymorphism
of the PPAR-γ2 gene.

Page 15

Advances in Therapy®
Anti-Obesity Effects of LG839
908
MTHFR
Di Renzo and associates have identi-
fied a subset of metabolically obese, but
normal weight individuals, with poten-
tially increased risks of developing the
metabolic syndrome, despite their normal
BMI.18 They determined the relationship
between body fat distribution, resting
metabolic rate, total body water amount,
and selected gene polymorphisms on inter-
leukin-15 receptor-alpha (IL-15Ralpha)
MTHFR C677T, to distinguish normal
weight obese (NWO) from non-obese
subjects with a normal metabolic profile
and obese individuals. The distribution of
MTHFR C677T and IL-15Ralpha geno-
types was significantly different between
classes. The data suggested that NWO in-
dividuals demonstrated a significant rela-
tionship between the decrease in the basal
metabolism, increased body fat mass, and
total body water amount. Possession of
wild type homozygote genotypes regard-
ing IL-15Ralpha cytokine and C677T
MTHFR enzyme characterized NWO in-
dividuals.18
Our results showing a negative cor-
relation between the MTHFR gene and
LG839 induction of weight loss is in agree-
ment with the Di Renzo study.
5-HT2A
While the serotonergic system and
receptors have been significantly associ-
ated with a number of eating disorders,49
to our knowledge this is the first report to
observe a negative association between the
–1438G/A allele of the 5-HT2A receptor
gene and resistance to a reduction in waist
circumference by administration of LG839.
DRD2
A Ser311Cys mutation of the human
DRD2 gene produces a marked functional
impairment of the receptor and is associ-
ated with higher BMI in some populations.
Tataranni hypothesized that the Ser311Cys
mutation of DRD2 may inhibit energy
expenditure.50 In a population of nondia-
betic Pima Indians, total energy expendi-
ture was lower in homozygotes for the
Cys311-encoding allele when compared
with those heterozygous and homozygous
for the Ser311-encoding allele (P=0.056).
The 24-hour resting energy expenditure was
also lower in homozygotes for the Cys311-
encoding allele when compared with those
heterozygous and homozygous for the
Ser311-encoding allele (P=0.026). These
findings were the first evidence that a genetic
mutation is associated with reduced energy
expenditure in humans. The findings of the
present study, whereby the TaqA1 allele was
significantly associated with a reduced num-
ber of exercise hours, provides additional
evidence that DRD2 gene polymorphisms
may influence both energy expenditure and
also actual exercise activity in a predisposed
individual. It is very interesting that of all the
genes tested, a positive correlation was ob-
tained with the DRD2 A1 allele and num-
ber of days in treatment. This is significant
because it would appear that those individu-
als carrying the DRD2 A1 allelic polymor-
phism continued treatment due to potential
benefits of LG839 targeted at a deficiency of
dopamine receptor (D2) density.