Download ppt - Fermata Pharma

Inhaled Buformin for
Lymphangioleiomyomatosis and
Lung Cancer
Repurposing an old drug
Steven Lehrer, MD
President
Fermata Pharma, Inc.
30 West 60th Street
New York, New York 10023-7909
[email protected]
Disclaimer
• The views and opinions expressed in the following PowerPoint
slides are those of the individual presenter and should not be
attributed to Drug Information Association, Inc. (“DIA”), its
directors, officers, employees, volunteers, members, chapters,
councils, Special Interest Area Communities or affiliates, or any
organization with which the presenter is employed or affiliated.
• These PowerPoint slides are the intellectual property of the
individual presenter and are protected under the copyright laws
of the United States of America and other countries. Used by
permission. All rights reserved. Drug Information Association,
Drug Information Association Inc., DIA and DIA logo are
registered trademarks. All other trademarks are the property of
their respective owners.
2
FERMATA Pharma
Inhaled Buformin for
Lymphangioleiomyomatosis and Lung
Cancer
Repurposing an old drug
Contributors
• John S. Patton, PhD
– Inventor of Exubera Inhaled Insulin, founder
of Nektar Therapeutics
• Peter H. Rheinstein, MD, MS, JD
– President, Severn Health Solutions
• James L. Mulshine, MD
– Associate Provost for Research, Rush
Medical University, Chicago
Fermata Summary– May 2016
• Development of buformin, a biguanide anti-diabetic and mTOR
inhibitor, to target Non- Small Cell Lung Cancer and
lymphangioleiomyomatosis, an orphan disease, via inhalation
• US patent 9,248,110 issued Feb 2, 2016
• Ample literature evidence shows that biguanides are associated with
a lower incidence of cancers in diabetics.
• mTor inhibition with oral sirolimus (rapamycin) is an effective
treatment for lymphangioleiomyomatosis; sirolimus(rapamycin) can
not be inhaled because of its lung toxicity (interstitial pneumonitis)
• Cancer medicines are the world’s top-selling drug category, with
$22.3 billion in US sales last year, up from $15.8 billion in 2006. FDA
approves new cancer drugs in just six months on average
• Orphan drug laws in the U.S. and Europe offer tax breaks and
market exclusivity for up to 10 years.
Lymphangioleiomyomatosis
• a rare lung disease caused by mTOR (mammalian
target of rapamycin) activation that results in a
proliferation of disorderly smooth muscle growth
(leiomyoma) throughout the lungs, resulting in the
obstruction of small airways, pulmonary cyst formation,
pneumothorax and lymphatic obstruction with chylous
pleural effusion.
• occurs in a sporadic form which only affects females
who are usually of childbearing age; also occurs in
patients who have tuberous sclerosis.
Lymphangioleiomyomatosis
Radiographic Appearance
• 23-year-old woman with
dyspnea, cough, hemoptysis,
and weight loss. Chest
radiograph demonstrates
normal-to-large lung volumes,
bilateral increased interstitial
opacities, and bilateral pleural
effusions (left one larger than
the right). The interstitial
opacities are more
pronounced in the lung
bases, and distinct cystic
structures are seen in the
right lung base (arrow).
Lymphangioleiomyomatosis
CT appearance
• Note the characteristic cysts throughout
the lung parenchyma in this young
woman.
mTOR
• The mammalian target of
rapamycin (mTOR) is a
serine/threonine protein
kinase that regulates cell
growth, cell proliferation,
cell motility, cell survival,
protein synthesis, and
transcription.
The mTOR (mammalian Target of
Rapamycin) Pathway
• mTOR integrates the input from
upstream pathways, including
insulin, growth factors (such as IGF1 and IGF-2), and amino acids.
mTOR also senses cellular nutrient
and energy levels. The mTOR
pathway is dysregulated in human
diseases, especially cancers.
Rapamycin is a bacterial product
that can inhibit mTOR via the
PI3K/AKT/mTOR pathway.
Rapamycin is toxic to the lung.
Biguanides can also inhibit mTOR
by AMPK activation but have no
lung toxicity.
Rapamycin inhibits mTOR
• Long-term treatment of mice and other organisms with rapamycin
extends life span and prevents tumors. But, at the same time,
rapamycin disrupts metabolic regulation and the action of insulin.
Lamming et al. dissected the action of rapamycin in genetically
modified mice and found that these two actions of rapamycin can be
separated. Rapamycin inhibits a protein kinase complex, mTORC1,
and this appears to provide most of the life-lengthening and antitumor effects of the drug. However, rapamycin also acts on a related
complex, mTORC2. The disruption of mTORC2 action produces the
diabetic-like symptoms of decreased glucose tolerance and
insensitivity to insulin.
• D. W. Lamming, L. Ye, P. Katajisto, M. D. Goncalves, M. Saitoh, D.
M. Stevens, J. G. Davis, A. B. Salmon, A. Richardson, R. S. Ahima,
D. A. Guertin, D. M. Sabatini, J. A. Baur, Rapamycin-induced insulin
resistance is mediated by mTORC2 loss and uncoupled from
longevity. Science 335, 1638–1643 (2012).
Biguanides inhibit only mTORC1
• Widely used generic class of small molecule drugs prescribed as
oral anti-diabetic
– Metformin
– Buformin (sold in Hungary, Rumania, Japan, and Taiwan)
– Phenformin withdrawn from US market because of its propensity
to cause lactic acidosis
– Benfosformin, etoformin, tiformin
• Well recognized as cell proliferation inhibitors
– AMPK activation and mTORC1 inhibition
– mTOR inhibitors are in development/use as anti-cancer agents
and for treatment of lymphangioleiomyomatosis
– Biguanides have no known lung toxicity after decades of use in
millions of patients
Lymphangioleiomyomatosis: Cause
and Treatment
• inappropriate activation of mammalian target of
rapamycin (mTOR) signaling, which regulates
cellular growth and lymphangiogenesis.
• Oral rapamycin (sirolimus), by inhibiting mTOR,
stabilizes lung function and is associated with a
reduction in symptoms and improvement in
quality of life.
• Rapamycin (Sirolimus) cannot be given inhaled
because of its lung toxicity (interstitial
pneumonitis).
Inhaled Buformin for
Lymphangioleiomyomatosis
• Oral metformin is being tested as treatment.
• The mTOR inhibitor buformin could be an ideal
inhaled treatment because it has no known lung
toxicity and can be given in inhaled doses that
may be one tenth or less of the toxic dose of
buformin required to produce lactic acidosis, its
principal side-effect.
FDA Approval of Rapamune, 2015
The safety and efficacy of Rapamune (rapamycin) for
treatment of LAM were studied in a clinical trial that compared
Rapamune with an inactive drug (placebo) in 89 patients for a
12-month treatment period, followed by a 12-month
observation period. The primary endpoint was the difference
between the groups in the rate of change in how much air a
person can exhale during a forced breath in one second
(forced expiratory volume in one second or FEV1). The
difference in the average decrease in FEV1 during the 12month treatment period was approximately 153 mL. After
discontinuation of Rapamune, the decline in lung function
resumed at a rate similar to the placebo group.
15
Lung Cancer – An Unmet Need
• 1.1 million new cases per year in men and 0.51
million new cases per year in women worldwide
• Cause of 28% of all cancer deaths in the US
– 85% NSCLC
• Cure rate 12% (16% 5 year survival)
• Current treatments
– Surgery followed by chemotherapy
– Expensive, very limited benefit
16% of lung cancer is Stage I
Localized at diagnosis
JNCI J Natl Cancer Inst (21 December 2005) 97(24): 1805
Lung Tumor Vulnerability
• Normal lung tissue is protected by cilia, which
beat constantly to move a protective layer of
fluid and mucous.
• Lung tumors have few or no cilia, little covering
of fluid, and so are vulnerable to inhaled
chemotherapy.
• Inhaled carboplatin, inhaled doxorubicin, and
inhaled temozolomide have been used for lung
cancer and are quite effective.
Lung Cancer and
Lymphangioleiomyomatosis–
Competitive Environment
• Lung cancer: Inhaled biguanides work by a different
mechanism than inhaled carboplatin, inhaled
doxorubicin, and inhaled temozolomide, and so would
be synergistic. In April 2010, Celgene invested $130
million in http://agios.com to discover and develop new
molecules that have the same action on tumors as
biguanides.
• Lymphangioleiomyomatosis responds to oral rapamycin
(sirolimus). But oral rapamycin is toxic to the lung,
causes interstitial pneumonitis, and could not be safely
administered by inhalation.
Cancer Cell Metabolism
Cancer cells and normal cells are metabolically
different. Ninety years ago Otto Warburg found
that while normal adult cells rely on mitochondrial
oxidative phosphorylation to generate energy,
cancer cells revert to a more primitive method of
metabolizing glucose, aerobic glycolysis,
fermenting glucose into lactate. Warburg
proposed that this phenomenon (now called the
Warburg effect) is an early step on the road to
cancer. The Warburg effect has been
demonstrated to occur across a wide spectrum of
tumors and is found in about 70% of cancers.
Warburg Effect
•
Rather than convert glucose to pyruvate and burn the pyruvate with oxygen
in the mitochondria, cancer cells convert the pyruvate to lactate in the
cytoplasm outside the mitochondria, and no oxygen is used. The process
yields only one-ninth the energy, four ATP molecules instead of 36, from
each molecule of glucose.
Oncogenic viruses enhance
glucose uptake
• affect cell proliferation pathways, such as
the PI3K and AMPK
– Rouse Sarcoma virus
– Kaposi’s sarcoma–associated herpes virus
(KSHV), the causative agent of Kaposi’s
sarcoma
– Human Papilloma Virus HPV
– SV40
– Hepatitis C Virus HCV
CT-PET Scanning
• Clinicians exploit
the Warburg effect
in tumor cells (the
increased rate of
glycolysis) for
detecting tumors by
18fluorodeoxyglucose
positron emission
tomography.
Warburg Effect and Insulin
• Insulin used in 1925 to disrupt Warburg effect
and shrink animal and human tumors
• Charles H Best, co-discoverer of insulin,
confirmed anti-tumor effect of insulin during
1950s.
• Last publication by Lilly Research Labs in 1959
confirmed earlier work and showed insulin +
glucagon was carcinostatic.
Warburg Effect and Biguanides
• To disrupt the Warburg effect, cancer
researchers are now investigating existing
drugs that are used in treating type 2
diabetes. Several studies have found that
an oral biguanide widely used for
diabetes, metformin, may help in
preventing cancer as well as in improving
outcomes when used with other cancer
therapies.
Biguanide Anti-Cancer effect
mechanism
• AMPK activation and mTOR inhibition
• Stimulation of tumor suppressor gene
LKB1
• lowering insulin and insulin-like growth
factor (IGF) levels. Metformin, for
example, decreases blood glucose, and,
as a secondary effect, decreases insulin
levels as well.
Metformin and Cancer I
• In diabetics who used metformin and comparators
(diabetics who had not used metformin):
• Lung cancer incidence
– 0.9% in diabetics on metformin
– 1.4% in comparators
• Mortality from cancer
– 3% in diabetics on metformin
– 6.1% in comparators
• The effect of metformin on cancer incidence and
mortality was significant (p < 0.001).
– Data from Libby G, Donnelly LA, Donnan PT, Alessi DR, Morris AD,
Evans JM (2009) New users of metformin are at low risk of incident
cancer: a cohort study among people with type 2 diabetes. Diabetes
Care 32: 1620-1625
Metformin and Cancer II
•
Kaplan-Meier plot with 95% CIs showing time to cancer among metformin users and
comparators. (from Libby G, Donnelly LA, Donnan PT, Alessi DR, Morris AD, Evans
JM (2009) New users of metformin are at low risk of incident cancer: a cohort study
among people with type 2 diabetes. Diabetes Care 32: 1620-1625)
Metformin and Lung Cancer
• Oral Metformin significantly improves chemotherapy outcomes and
survival for patients who have non-small cell lung cancer with
diabetes.
The Overall survival for Group A (metformin) was significantly longer than that for Group B (insulin; P = 0.034) and
Group C (other drugs; P = 0.003). (Tan BX, Yao WX, Ge J, Peng XC, Du XB, Zhang R, Yao B, Xie K, Li LH, Dong
H, Gao F, Zhao F, Hou JM, Su JM, Liu JY. Prognostic influence of metformin as first-line chemotherapy for
advanced non-small cell lung cancer in patients with type 2 diabetes. Cancer. 2011 Apr 26. doi:
10.1002/cncr.26151. [Epub ahead of print])
Biguanide delivery mode
• Inhalation aerosol is the preferred route of administration
– Limits risk of systemic side effects associated with biguanides
because of low inhaled dose
• Lactic acidosis is main systemic side effect
– More predictable dose to the lung
– Established mode of delivery for a range of therapeutic agents
– Precedence exists for use
• Cytotoxic agents have been tested via this route specifically as
lung cancer therapy
• Various animal studies and clinical case report studies have
been conducted
• Biguanides should have a long lung residence time
Inhaled and oral dosing
• Typical inhaled asthma medications are about
10-100 µg day, inhaled insulin requires 1-10 mg
per day.
• Metformin is taken orally twice daily by diabetics,
maximum total dose 2.5 g/day.
• This oral dose is associated with reduced
cancer incidence but higher doses cannot be
used because of a serious side effect, lactic
acidosis.
Inhaled Metformin disadvantage
• Reducing the inhaled dose of an oral drug by a factor of
10-20 typically results in the same local concentration in
the airways as by oral administration. So just to equal
what the oral dose of metformin would "deliver" to the
airways, a subject would need to inhale 125-250 mgs
metformin per day; or, if broken into 3 doses a day, 4080 mg/dose.
• Delivering this much metformin powder to the lung is at
the upper limit of acceptability, and would result in
reduced compliance, bronchospasm and cough.
Inhaled Buformin advantage
• Buformin has eight times the potency of metformin. Using inhaled
buformin as opposed to metformin, one could reduce the dose by a
factor of eight. The usual maximum oral dose of buformin is 300 mg
per day. Dropping the inhaled dose by a factor of 10 to 20, three
doses per day inhaled buformin could be given at 5mg to 10 mg per
dose, much less than metformin.
• This dose of buformin, 15mg to 30mg per day, would be highly
unlikely to produce lactic acidosis, the main biguanide complication.
In one report, the toxic oral buformin dose was 329 ± 30mg/day in
24 patients who developed lactic acidosis on buformin. Another
group of 24 patients on 258 ± 25mg/day buformin did not develop
lactic acidosis.
• In other words, one can increase the inhaled buformin dose ten
times above what would be needed to treat lung cancer or
lymphangioleiomyomatosis and still be well below the systemic toxic
dose. This is a key strength of buformin.
Buformin Dosage
400
Mg
300
200
100
0
INHALED
THERAPEUTIC
BUFORMIN DOSE
TOXIC
Long buformin lung residence time
• Buformin has an octanol/water partition
coefficient (log P) of -1.2 and is
hydrophilic. Hydrophilic small molecules
with a log P less than 0 have a mean lung
half life (t½) of about one hour.
• Long lung residence time of buformin
could be increased even further with an
appropriate formulation.
•
John S. Patton, C. Simone Fishburn, and Jeffry G. Weers. The Lungs as a Portal of Entry for Systemic Drug
Delivery. Proc Am Thorac Soc Vol 1. pp 338–344, 2004.
Buformin – Competitive Advantages
• Repurposed drug with known systemic
safety profile
• Well established Chemistry, Manufacturing
and Control profile
• Inexpensive supply with Drug Master Files
on file
• Non-toxic compared to vast majority of anticancer agents
• Readily administered by inhalation aerosol
Fermata Pharma – Intellectual Property
• Broad claims filed on use of biguanides in
lung cancer and lymphangioleiomyomatosis
– US Patent 9,248,110, Compositions and Methods
of Treating and Preventing Lung Cancer and
Lymphangioleiomyomatosis, issued February 2,
2016
Risks
• Does buformin exhibit adequate potency?
• Is buformin rapidly and completely absorbed from
the lungs?
– Would diminish targeting benefit of inhaled
therapy
– Would increase risk of systemic side effects
• Formulation?
– Risks lessened through appropriate formulation
– Potential use of Dry Powder Inhaler
• Unacceptable pulmonary toxicology?
• Emerging novel therapies?
Inhaled Buformin: Obstacles
• As John Lisman has already noted:
• It is expensive to create the necessary
dossier for a marketing authorization
• Investment in clinical evidence of any new
application of old active substances can
seem to be a waste of money
• Regulatory systems do not allow easy
access for new indications for old active
substances
Other uses of inhaled buformin
• We will seek FDA approval for only the
narrowest uses of inhaled buformin:
treatment of lymphangioleiomyomatosis
and stage I localized lung cancer. But we
will later seek approval for other uses also
covered by our IP:
– Treatment of all stages of lung cancer
– Tumor radiation sensitization in lung cancer patients undergoing
radiotherapy
– Prevention of lung cancer in heavy smokers
– Treatment of lung metastases
Alternate Formulations
• For lung cancer treatment and prevention, it
could be desirable to have a buformin
formulation and special inhaler that produce an
aerosol mimicking the distribution of cigarette
and cigar smoke in the lung.
• This refinement might not be necessary because
of the high dose of buformin that can be
delivered to the lung by inhalation without
systemic toxicity.
Inhaled buformin clinical trial
• FDA will probably allow human testing
of inhaled buformin in lung cancer
patients after animal PK and
toxicology studies. Biguanides and
buformin in particular have no lung
toxicity after decades of use in millions
of patients.
Timetable and Expenditures
Valuation and Exits
Phase II
Prelim Efficacy?
Finalize delivery mode/dose
Value
Phase I
Tolerability
Patent
Issue
Toxicology
POC
Initial
$ infusion
2nd
$ infusion
Tranche, Grant?
3rd
$ infusion
Cost/Time
Exit point
10x ROI?
Clinical Trials
•
The clinical program includes three clinical studies: two dose ranging
studies and one two year safety and efficacy study. Each of the studies is
briefly described below.
– Phase one is a randomized, double-blind, double-dummy, placebo-controlled,
two-period, crossover, study of 3 daily doses daily of buformin inhalation aerosol
and placebo administered via inhaler to 20 normal adults. A double-dummy
design allows additional insurance against bias or placebo effect. All patients are
given both placebo and active doses in alternating periods of time during the
study.
– Phase two is a randomized, double-blind, double-dummy, placebo-controlled,
two-period, crossover, study of 20 daily doses of buformin inhalation aerosol and
placebo administered via inhaler to 20 adults with localized lung cancer.
– Phase three is a two year, randomized, double-blind, placebo controlled, parallel
group safety and survival study of buformin inhalation aerosol and placebo
administered via inhaler. A total of 199 stage I (localized lung cancer) patients
will enter this study. The probability is 80 percent that the study will detect a
treatment difference at a two sided 5% significance level, if there is 40% survival
in the placebo group and 80% survival in the buformin group. This is based on
the assumption that the accrual period will be 24 Months and the median survival
is 13 Months. The total number of deaths will be 65.
Total Cost
• At $10,000 per patient, plus cost of
formulating buformin aerosol, manufacture
of inhalers, animal toxicity and dosage
studies in one rodent and one non-rodent
species to obtain IND, total cost of study:
• $3.9 million
Summary
Our mission is to provide a new form of chemotherapy for lung cancer and
lymphangioleiomyomatosis: an inhaled biguanide, buformin. This drug, used to treat
type II diabetes, has anticancer activity and could be delivered by inhalation to a lung
tumor in much higher concentrations than can be administered orally. Another
biguanide, oral metformin, is now the most widely prescribed anti-diabetic drug in the
world; in the United States alone, more than 40 million prescriptions were filled in
2008 for its generic formulations. Biguanide anti-diabetic agents inhibit cancer by
changing the metabolism of cancer cells, which are metabolically different from
normal cells. Several studies have found that metformin and other biguanides in vitro
and in vivo may help in preventing cancer as well as in improving outcomes when
used with standard cancer therapies. Cancer of the lung and
lymphangioleiomyomatosis could be very amenable to treatment with inhaled
buformin. When administered as an aerosol by inhalation, buformin could infiltrate
lung tissue in much higher concentrations than could be achieved orally, rendering
the pathologic cells much more vulnerable to treatment. Inhaled buformin would be
synergistic with other forms of chemotherapy. Inhaled buformin could also be used to
prevent lung cancer in high risk individuals.
References
•Algire C, Zakikhani M, Blouin MJ, Shuai JH, Pollak M (2008) Metformin attenuates the stimulatory effect of a high-energy diet on in vivo LLC1
carcinoma growth. Endocr Relat Cancer 15: 833-839
•Antonoff MB, D'Cunha J. Teaching an old drug new tricks: metformin as a targeted therapy for lung cancer. Semin Thorac Cardiovasc Surg
2010;22(3):195-6.
•Ben Sahra I, Le Marchand-Brustel Y, Tanti JF, Bost Fdr (2010) Metformin in Cancer Therapy: A New Perspective for an Old Antidiabetic Drug?
Molecular Cancer Therapeutics 9: 1092-1099
•Carvalho TC, Carvalho SR, McConville JT. Formulations for pulmonary administration of anticancer agents to treat lung malignancies. J Aerosol
Med Pulm Drug Deliv 2011 Apr;24(2):61-80.
•Friedrich MJ (2010) Researchers aim to stop tumor growth by shutting off cancer's fuel supply. JAMA 303: 1021-1022
•Gagnadoux F, Pape AL, Lemarie E, Lerondel S, Valo I, Leblond V, Racineux JL, Urban T (2005) Aerosol delivery of chemotherapy in an orthotopic
model of lung cancer. Eur Respir J 26: 657-661
•Govindarajan R, Ratnasinghe L, Simmons DL, Siegel ER, Midathada MV, Kim L, Kim PJ, Owens RJ, Lang NP (2007) Thiazolidinediones and the
risk of lung, prostate, and colon cancer in patients with diabetes. J Clin Oncol 25: 1476
•Landman GW, Kleefstra N, Van Hateren KJ, Groenier KH, Gans RO, Bilo HJ (2010) Metformin associated with lower cancer mortality in type 2
diabetes: ZODIAC-16. Diabetes Care 33: 322-326
•Libby G, Donnelly LA, Donnan PT, Alessi DR, Morris AD, Evans JM (2009) New users of metformin are at low risk of incident cancer: a cohort study
among people with type 2 diabetes. Diabetes Care 32: 1620-1625
•Lin, C. C., Shen, M. C., and Su, W. C. Metformin inhibits lung cancer cell growth and angiogenesis in vitro and in vivo: Effect on IL6-Stat3 pathway.
Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC 51, 19. 2010.
•Memmott R, Mercado J, Maier C, Kawabata S, Fox S, Dennis P (2010) Metformin Prevents Tobacco Carcinogen-Induced Lung Tumorigenesis.
Cancer Prevention Research 3: 1066-1076
•Monami M, Lamanna C, Balzi D, Marchionni N, Mannucci E (2009) Sulphonylureas and cancer: a case-control study. Acta Diabetol 46: 279-284
•Onn A, Isobe T, Itasaka S, Wu W, O'Reilly MS, Ki Hong W, Fidler IJ, Herbst RS (2003) Development of an Orthotopic Model to Study the Biology
and Therapy of Primary Human Lung Cancer in Nude Mice. Clin Cancer Res 9: 5532-5539
•Otterson GA, Villalona-Calero MA, Hicks W, Pan X, Ellerton JA, Gettinger SN, Murren JR (2010) Phase I/II Study of Inhaled Doxorubicin Combined
with Platinum-Based Therapy for Advanced Non-Small Cell Lung Cancer. Clin Cancer Res 16: 2466-2473
•Tan BX, Yao WX, Ge J, Peng XC, Du XB, Zhang R, Yao B, Xie K, Li LH, Dong H, Gao F, Zhao F, Hou JM, Su JM, Liu JY. Prognostic influence of
metformin as first-line chemotherapy for advanced non-small cell lung cancer in patients with type 2 diabetes. Cancer. 2011 Apr 26. doi:
10.1002/cncr.26151. [Epub ahead of print]
•Wauthoz N, Deleuze P, Hecq J, Roland I, Saussez S, Adanja I, Debeir O, Decaestecker C, Mathieu V, Kiss R, Amighi K (2010) In vivo assessment
of temozolomide local delivery for lung cancer inhalation therapy. Eur J Pharm Sci 39: 402-411
•Yi D, Wiedmann TS (2010) Inhalation Adjuvant Therapy for Lung Cancer. J Aerosol Med Pulm Drug Deliv
•Zarogoulidis, P., Eleftheriadou, V., Zarogoulidou, I., Sapardanis, I., Kontakiotis, T., Sichletidis, L., and Zarogoulidis, K. Feasibility and effectiveness
of inhaled carboplatin in the treatment of NSCLC patients. Journal of Clinical Oncology 28[suppl], e17511. 2010.