FROM PORINS TO VAPTANS :A LOT OF WATER HAS FLOWN

DR. AYUSMATI THAKURMBBS, DNB (GENERAL MEDICINE)

The Physiology –Aquaporins and Vasopressin

Background

Every cell is primarily water.

But the water doesn’t just sit in the cell, it moves through it in a very organized way.

For many years, scientists assumed that water leaked through the cell membrane, and some water does.

"But the very rapid movement of water through some cells was not explained by this theory," said Agre.

Aquaporins

The discovery of aquaporin-1 answered the long-standing biophysical question of how water specifically crosses biological membranes.

Agre discovered aquaporins "by serendipity." The Rh molecule was isolated but a second molecule, 28 kDa in size kept appearing, which turned out to be an undiscovered molecule with unknown function. It was abundant in RBC and kidney tubes, and related to proteins of diverse origins, like the brains of fruit flies, bacteria, the lenses of eyes, and plant tissues.

The 2003 Nobel Prize in Chemistry was awarded jointly to Peter Agre for the

discovery of aquaporins

• Aquaporins are integral membrane proteins from a larger family of major intrinsic proteins (MIP) that form pores in the membrane of biological cells.

• Aquaporins are "the plumbing system for cells“• Aquaporins selectively conduct water molecules in and out of the cell, while

preventing the passage of ions and other solutes. • Aquaporins form tetramers in the cell membrane, with each monomer acting as a

water channel

Body water balance is tightly regulated by vasopressin, and multiple studies now have underscored the essential roles of AQP2 in this.

Vasopressin regulates acutely the water permeability of the kidney collecting duct by trafficking of AQP2 from intracellular vesicles to the apical plasma membrane.

In the kidney, at least seven aquaporins are expressed at distinct sites.

AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. 2

AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel.

AQP3 and AQP4 are both present in the baso-lateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have demonstrated that both AQP2 and AQP3 are essential for urinary concentration.

Three additional aquaporins are present in the kidney.

AQP6 is present in intracellular vesicles in collecting duct intercalated cells, and AQP8 is present intracellularly at low abundance in proximal tubules and collecting duct principal cells, but the physiological function of these two channels remains undefined.

AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption.

“If aquaporin could be manipulated, that could potentially solve medical problems such as fluid retention in heart disease and brain edema after stroke,” said Agre.

Mutations in the aquaporin-2 gene cause hereditary nephrogenic diabetes insipidus in humans.

Mice homozygous for inactivating mutations in the aquaporin-0 gene develop congenital cataracts

It has been found that autoimmune reactions against aquaporin 4 produce Devic's disease

VASOPRESSIN

• Arginine vasopressin (AVP), also known as vasopressin or antidiuretic hormone (ADH), is a neurohypophysial hormone found in most mammals.

• The Vasopressins are peptides consisting of nine amino acids (nonapeptides).

• Two primary functions - to retain water in the body and to constrict blood vessels.

• One of the most important roles of AVP is to regulate the body's retention of water. In normal conditions it is released when the body is dehydrated and causes the kidneys to conserve water, thus concentrating the urine and reducing urine volume.

• At high concentrations, it also raises blood pressure by inducing moderate vasoconstriction.

Supra-optic and Paraventricular

nucleus

PHYSIOLOGY

ControlVasopressin is secreted from the posterior pituitary gland in response to reductions in plasma volume, in response to increases in the plasma osmolality, and in response to cholecystokinin(CCK) secreted by the small intestine:• Secretion in response to reduced plasma volume is activated by pressure

receptors in the veins, atria and carotids.• Secretion in response to increases in plasma osmotic pressure is mediated

by osmoreceptors in the hypothalamus.• Secretion in response to increases in plasma CCK is mediated by an unknown

pathway.

Many factors influence the secretion of vasopressin:• Ethanol reduces the calcium-dependent secretion of AVP by blocking voltage-

gated calcium channels in neurohypophyseal nerve terminals.• Angiotensin II stimulates AVP secretion, in keeping with its general pressor and

pro-volumic effects on the body.• Atrial natriuretic peptide inhibits AVP secretion, in part by inhibiting

Angiotensin II-induced stimulation of AVP secretion.

VASOPRESSIN RECEPTOR

RECEPTOR SIGNALLING PATHWAY LOCATION FUNCTION

V1A G protein-coupled, phosphatidylinositol/calcium

Vascular smooth muscle, platelet, hepatocytes,

myometrium

Vasoconstriction, myocardial hypertrophy,

platelet aggregation, glycogenolysis, uterine

contraction

V1B G protein-coupled, phosphatidylinositol/calcium

Anterior pituitary gland Releases ACTH, prolactin, endorphins

V2 Adenylyl cyclase/ cAMPBasolateral membrane

of CD, Vascular endothelium and

vascular smooth muscle cell

Insertion of AQP-2 water channels into apical

membraneInduction of AQP-2

synthesisReleases von Willebrand

factor and factor VIII, Vasodilation

There are three subtypes of vasopressin receptor: V1A (V1), V1B (V3) and V2

VASOPRESSIN RECEPTOR ANTAGONIST

A vasopressin receptor antagonist (VRA) is an agent which interferes with action at the vasopressin receptors.

A new class of medication, the "vaptan" drugs, act by inhibiting the action of vasopressin on its receptors (V1A, V1B and V2).

The vaptan class of drugs contains a number of compounds with varying selectivity, several of which are either already in clinical use or in clinical trials.

CLASS OF RECEPTOR BLOCK NAME

Unselective (mixed V1A/V2) CONIVAPTAN

V1A selective (V1RA) RELCOVAPTAN

V1B selective (V3RA) NELIVAPTAN

V2 selective (V2RA)LIXIVAPTAN

MOZAVAPTANSATAVAPTANTOLVAPTAN

CLASSIFICATION

Potential uses of Vasopressin Receptor Antagonists

Uses of Vasopressin Receptor Antagonists

• Hyponatremia Euvolemic :• SIADH Hypervolemic – • Congestive Cardiac Failure• Cirrhosis

• Polycystic Kidney Disease

• Hyponatremia Euvolemic :

• SIADH Hypervolemic – • Congestive Cardiac Failure• Cirrhosis

• Polycystic Kidney Disease

• V2R antagonists have become a mainstay of treatment for : - • Euvolemic (i.e., SIADH, postoperative hyponatremia)

Hypervolemic hyponatremia (i.e., CHF and cirrhosis).• V2RAs predictably cause aquaresis leading to increased [Na+]

in majority of patients with hyponatremia due to SIADH, CHF, and cirrhosis.

• For hyponatremia in hospitalized patients, who are unable to take medication orally or for those in whom a more rapid correction of hyponatremia is desired, conivaptan (V1/V2R antagonist) will likely be the preferred agent.

• Selective V2R antagonists such as tolvaptan, lixivaptan etc. will likely be useful in patients for whom oral therapy is suitable and for more chronic forms of hyponatremia.

Selective vasopressin V2-receptor antagonists, also called ‘vaptans’, represent the first targeted therapy for SIADH, one of the most common causes of hyponatraemia

Tolvaptan was approved by the European Medicines Agency for the treatment of hyponatraemia secondary to SIADH.

It was proposed that SIADH should be called the syndrome of inappropriate antidiuresis (SIAD), because vasopressin (antidiuretic hormone) is not always elevated, for example in patients with reset osmostat or with an activating mutation of the vasopressin V2-receptor.

Types of the Syndrome of Inappropriate Antidiuresis (SIAD).

Ellison DH, Berl T. N Engl J Med 2007;356:2064-2072.

Types of SIAD. Patterns of plasma levels of arginine vasopressin (AVP); as compared with plasma Na levels in patients with SIAD, are shown. Type A is characterized by unregulated secretion of AVP, type B by elevated basal secretion of AVP despite normal regulation by osmolality,Type C by a “reset osmostat,” and type D by undetectable AVP. The shaded area represents normal values of plasma AVP.

Causes of the SIADH

Ellison DH, Berl T. N Engl J Med 2007;356:2064-2072.

Mozavaptan has been approved in Japan for paraneoplastic SIADH.

The European Medicines Agency (EMEA) has approved tolvaptan specifically for the treatment of adult patients with hyponatremia secondary to SIADH.

Conivaptan has been used in dilutional hyponatremia associated with hypothyroidism, renal dysfunction, and malignancies.

Conivaptan (Vaprisol) Approved for Euvolemic Hyponatremia in Hospitalized Patients

On December 29, 2005, the FDA approved Conivaptan HCl injection (Vaprisol) for the treatment of euvolemic hyponatremia (eg, the syndrome of inappropriate secretion of antidiuretic hormone or in the setting of

hypothyroidism, adrenal insufficiency, pulmonary disorders, etc) in hospitalized patients.

FDA Approves Tolvaptan (Samsca), the First and Only Oral Vasopressin Antagonist to Treat Patients With Clinically Significant Hypervolemic and Euvolemic Hyponatremia

First approved May 19th, 2009

Verbalis JG, Goldsmith SR, Greenberg A, Schrier RW, Sterns H. Hyponatremia treatment guidelines 2007: Expert panel recommendations. Am J Med 2007;120:S1-21.

A recent publication indicated that severe acute hyponatremia (<118 mmol/L) might become an indication for vaptan treatment, citing two reasons:

- Greater ease in terms of titrating the correction rate of hyponatremia with vaptan than with hypertonic saline.

- No risk of pulmonary edema in response to vaptan as opposed to hypertonic saline.

• Hyponatremia Euvolemic :• SIADH Hypervolemic –

• Congestive Cardiac Failure• Cirrhosis

• Polycystic Kidney Disease

It is well established that the neurohormonal activation characteristic of CHF, including increased renin, angiotensin, aldosterone, and catecholamines, contributes to progression of CHF.

Abundant evidence from large-scale clinical trials attests to the reduction in cardiovascular mortality that accrues from treatment with angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, adrenergic blockers, and aldosterone antagonists.

Subgroup analysis suggests that cardiovascular mortality may be reduced by the selective V2R antagonist tolvaptan in the higher risk group with kidney function impairment or severe congestive findings.

As the V1aR is responsible for the pressor and mitogenic effects of AVP and as plasma AVP levels rise in some settings during V2R blockade, it is also possible that long-term isolated V2R blockade could prove deleterious to patients with CHF.

A well-powered study to determine whether selective V2R antagonist therapy will prove as beneficial as other inhibitors of the CHF neurohormonal cascade is ongoing, although it will not establish whether long-term combined V1a and V2R antagonism might be superior.

Until such studies have been completed and an FDA indication is granted for use in CHF with or without accompanying hyponatremia, VRAs are not recommended in patients with CHF.

• Hyponatremia Euvolemic :• SIADH Hypervolemic – • Congestive Cardiac Failure

• Cirrhosis

• Polycystic Kidney Disease

Although the hyponatremia of cirrhosis is analogous to that of CHF in many respects, the presence of portal hypertension in these patients presents additional concerns, namely, that V1aR blockade may produce hypotension and variceal bleeding owing to antagonism of AVP pressor effects in this vascular bed.

Consequently, at the present time, only selective V2R antagonists can be recommended for these patients.

The lixivaptan and tolvaptan trials provide conflicting data as to whether cirrhotic patients will be as responsive as patients with other causes of hyponatremia.

TRIALS

The trials

Studies of Ascending Levels of Tolvaptan (SALT-1 and SALT-2) were designed to focus specifically on changes in serum Na in patients with hyponatremia from multiple disorders including SIADH, heart failure, and cirrhosis.

The studies enrolled 205 and 243 patients, respectively. The patients received daily oral doses of either placebo or tolvaptan 15 mg with titration to 30 and 60 mg four times a day (as needed) to correct sodium over 30 days, with a follow-up visit seven days after the end of the study.

Primary endpoints were average change in serum sodium from baseline to day 4 and day 30 of treatment.

In SALT-1, patients receiving tolvaptan had an average daily area AUC change in serum Na by day 4 of 3.62 ± 2.68 mEq/L as compared to 0.25 ± 2.08 mEq/L in the placebo group (P < 0.0001). At day 30, this averaged 6.22 ± 4.10 for tolvaptan and 1.66 ± 3.59 mEq/L for placebo (P < 0.0001). The results of SALT-2 were similar. In both studies, serum sodium improved more in the tolvaptan-treated patients. The tolvaptan group of patients required less restriction of fluids and it was superior to placebo in raising and maintaining serum sodium concentration.

However, during the seven-day follow-up period (after stopping tolvaptan), hyponatremia was again observed, indicating that the continued aquaretic effect of tolvaptan was required to maintain normal sodium concentrations in patients with chronic hyponatremia, although long-term studies do not support this.

Safety and sodium Assessment of Long-term Tolvaptan With hyponatremia:

A year-long, open-label Trial to gain Experience under Real-world conditions (SALTWATER) was an open-label extension of the earlier SALT study in which the SALT enrollees who previously received either tolvaptan or placebo for 30 days were given oral tolvaptan for up to 804 days.

A total of 111 individuals participated in SALTWATER, of whom 64 discontinued the drug, 30 because of death or adverse reactions.

At 50 weeks, the serum sodium concentration normalized in approximately 60% of the patients.

- Six placebo-controlled clinical trials of vaptan therapy included patients with congestive

heart failure or cirrhosis, as well as individuals with euvolemic hyponatremia.

- All of these studies found that vaptans produced variable increases in urine output and

plasma sodium levels in the group as a whole, and some noted a modest improvement

in mental status or signs and symptoms of congestive heart failure.

- However, no improvement in long-term mortality or re-hospitalization rates was

demonstrated. Apart from symptoms of thirst and polyuria, as well as occasional

asthenia and constipation, no drug-related adverse events were noted, even though the

rise in serum sodium levels in a few patients exceeded the limit recommended to avoid

the risk of osmotic demyelization.

- None of these studies separated the findings in patients with hypervolemic

hyponatremia from those with other types of hyponatremia. Thus, they do not permit

conclusions about possible differences in efficacy or safety that result from differences in

the pathophysiology of impaired water excretion.

1. Ghali, J. K. et al. Efficacy and safety of oral conivaptan: a V1A/V2 vasopressin receptor antagonist, assessed in a randomized, placebo-controlled trial in patients with euvolemic or hypervolemic hyponatremia. J. Clin. Endocrinol. Metab. 91, 2145-2152 (2006).

2. Annane, D., Decaux, G. & Smith, N. Efficacy and safety of oral conivaptan, a vasopressin-receptor antagonist, evaluated in a randomized, controlled trial in patients with euvolemic or hypervolemic hyponatremia. Am. J. Med. Sci. 337, 28-36 (2009).

3. Zeltser, D., Rosansky, S., van Rensburg, H., Verbalis, J. G. & Smith, N. Assessment of the efficacy and safety of intravenous conivaptan in euvolemic and hypervolemic hyponatremia. Am. J. Nephrol. 27, 447-457 (2007).

4. Schrier, R. W. et al. Tolvaptan, a selective oral vasopressin V2-receptor antagonist, for hyponatremia. N. Engl. J. Med. 355, 2099-2112 (2006).

5. Gheorghiade, M. et al. Vasopressin v(2) receptor blockade with tolvaptan versus fluid restriction in the treatment of hyponatremia. Am. J. Cardiol. 97, 1064-1067 (2006).

6. Wong, F., Blei, A. T., Blendis, L. M. & Thuluvath, P J. A vasopressin receptor antagonist (VPA-985) improves serum sodium concentration in patients with hyponatremia: a multicenter, randomized, placebo-controlled trial. Hepatology 37, 182-191 (2003).

Patients who fulfilled the criteria for euvolemic hyponatremia were included with others who had hypervolemic hyponatremia in five placebo-controlled trials of

vaptan therapy.

However, the results were pooled and reported without distinction as to the type of hyponatremia

Two other trials focused exclusively on patients with euvolemic hyponatremia. The patients were categorized as having SIADH, but the proportion of individuals who may have had other vasopressin-dependent or vasopressin-independent causes of inappropriate antidiuresis could not be determined from the data provided.

O'Leary, J. G. & Davis, G. L. Conivaptan increases serum sodium in hyponatremic patients with end-stage liver disease. Liver Transpl. 15, 1325-1329 (2009).

Soupart, A. et al. Successful long-term treatment of hyponatremia in syndrome of inappropriate antidiuretic hormone secretion with satavaptan (SR121463B), an orally active nonpeptide vasopressin V2-receptor antagonist. Clin. J. Am. Soc. Nephrol. 1,1154-1160 (2006).

Four other reports focused exclusively on patients with congestive heart failure.

These studies were concerned primarily with the effect of vaptans on hemodynamic function and included few patients with hyponatremia.

Nevertheless, they provide useful information about the effects of vaptans on water balance and plasma sodium levels in congestive cardiac failure.

Gheorghiade, M. et al. Vasopressin V2-receptor blockade with tolvaptan in patients with chronic heart failure: results from a double-blind, randomized trial. Circulation 107,2690-2696 (2003).

This study compared the effects of placebo and three different doses of tolvaptan for up to 23 days in 254 patients.

A second study evaluated the acute effects of five different doses (10-400 mg) of lixivaptan in patients with NYHA class III congestive heart failure and normal or low serum sodium levels.This study found that, compared with placebo, doses of lixivaptan >10 mg produced variable increases in urine flow and free water clearance, 2-4 h after treatment. At higher doses (150-400 mg), the increased water excretion was associated with a statistically significant rise in levels of serum sodium (of unspecified magnitude) and plasma vasopressin. The effect on hyponatremic symptoms, if any, was not reported. No serious adverse events were linked specifically to the treatment.

Abraham, W. T., Shamshirsaz, A. A., McFann, K., Oren, R. M. & Schrier, R. W. Aquaretic effect of lixivaptan, an oral, non-peptide, selective V2 receptor vasopressin antagonist, in New York Heart Association functional class II and III chronic heart failure patients. J. Am. Coll. Cardiol. 47, 1615-1621 (2006).

Two additional studies were concerned mainly with the effects of tolvaptan on cardiac

function and survival in patients with congestive heart failure. In one study, 45 of 68

hospitalized patients with hyponatremia increased their plasma sodium by at least 2 mmol/l

during treatment with 30 mg, 60 mg or 90 mg doses of tolvaptan. These patients also had a

lower mortality rate (11%) 60 days after discharge than did patients whose hyponatremia

did not improve (22%). This finding suggests that correction of hyponatremia in congestive

failure improves the poor outcome with which it is otherwise associated. Thus, the major

benefits of vaptan therapy seem to be limited to patients with hyponatremia.

Rossi, J. et al. Improvement in hyponatremia during hospitalization for worsening heart failure is associated with improved outcomes: insights from the Acute and Chronic Therapeutic Impact of a Vasopressin Antagonist in Chronic Heart Failure (ACTIV in CHF) trial. Acute Card. Care 9, 82-86 (2007).

Short-term trials:

EVEREST (Efficacy of Vasopressin antagonist in hEart FailuRE outcome Study with Tolvaptan)

ACTIV in CHF (Acute and Chronic Therapeutic Impact of Vasopressin antagonist in Congestive Heart Failure)

Showed a rapid increase in serum sodium secondary to increase in urine output but despite showing improvement in hemodynamic parameters like pulmonary capillary wedge pressure (PCWP)-a clinical indicator of preload, improvement in clinical status was not significant, reflecting that improvement in hyponatremia is not an index of improvement in clinical outcome.

Similarly, long-term trials have failed to demonstrate a favorable effect on morbidity and mortality.

Moreover, strict monitoring of serum sodium is discomforting for the patient.

• Hyponatremia Euvolemic :• SIADH Hypervolemic – • Congestive Cardiac Failure• Cirrhosis

• Polycystic Kidney Disease

• Hyponatremia

• Congestive Cardiac Failure

• Cirrhosis

• Polycystic Kidney Disease

BACKGROUND

• Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disease and the fourth leading cause of ESRD in adults worldwide.

• It results in the progressive development of kidney cysts, kidney pain, hypertension, and, ultimately, kidney failure. Effective treatment for ADPKD has been lacking.

• Studies of animal models implicate the antidiuretic hormone AVP and its second messenger c-AMP as promoters of kidney-cyst cell proliferation and luminal fluid secretion.

• The suppression of vasopressin release by means of high water intake, genetic elimination of vasopressin, and vasopressin V2-receptor blockade all reduce the cyst burden and protect kidney function.

• Such preclinical studies justify studying the effects of Tolvaptan, a vasopressin V2-receptor antagonist, in human ADPKD.

Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and Its Outcomes (TEMPO) trial

Results and conclusions

Adverse Effects

The safety profile of the vaptans is good with no consistent adverse effects other than thirst.

Intravenous Conivaptan can cause inflammation at infusion sites .

Tolvaptan has been associated with an increased incidence of gastrointestinal bleeding in patients with liver disease and should be used with caution and only if absolutely necessary in patients with this condition.

Occasionally, vaptans have been observed to raise plasma sodium levels faster or higher than is recommended to avoid osmotic demyelination, but this potential complication was not observed in any trial reported to date. Nevertheless, concern over this effect has led some clinicians to advise against the use of fluid restriction with vaptan therapy, at least during the first 24 h, even though thirst is a common adverse effect and increased fluid intake may offset much of the desired effect on plasma sodium levels.

In any case, the effects on plasma sodium concentration should be monitored closely during treatment and used to guide adjustments to fluid intake and continued dosing.

Current Status of Vaptans

Vaptans are the most appropriate physiological approach to treat hyponatremia as they do not deplete electrolytes and can spare the stringent and inconvenient restriction of fluids.They do not stimulate the neurohormonal system and cause no renal impairment. Vaptans can help to decrease the dose of diuretics in CHF.

However a lack of consistency for correction of serum sodium by vaptans has been observed in trials. The follow-up treatment is not well defined. Its property of inhibition of CYP can cause drug-drug interaction. Despite weaker interactions, tolvaptan is still contraindicated with strong CYP3A4 inhibitors and the drug may be less effective when used with potent CYP3A4 inducers such as rifampicin. Reductions in dose should be considered when tolvaptan is used with P-glycoprotein inhibitors such as cyclosporine. Dual blockers (V 1 a/V 2 ) may cause bleeding complications.Studies in the pediatric population are also lacking. Their high cost is another factor to be considered.

Vaptans should not be used to treat the type of euvolemic hyponatremia caused by emetic stimuli or secondary adrenal insufficiency, and they are ineffective in the vasopressin-independent form of SIADH (caused by an activating mutation of the V 2 receptor). They are ineffective where AVP levels are appropriate, for example, cerebral salt wasting and psychogenic polydipsia. Moreover, their effect on the vascular endothelium is unknown.

Samsca (Tolvaptan): Drug Warning - Potential Risk of Liver Injury

FDA ALERT

ISSUE: Otsuka and FDA notified healthcare professionals of significant liver injury associated with the use of Samsca.

Most of the liver enzyme abnormalities were observed during the first 18 months of therapy. Following discontinuation of treatment, all patients improved.

RECOMMENDATION: Healthcare providers should perform liver tests promptly in patients who report symptoms that may indicate liver injury, including fatigue, anorexia, right upper abdominal discomfort, dark urine or jaundice. If hepatic injury is suspected, Samsca should be promptly discontinued, appropriate treatment should be instituted, and investigations should be performed to determine probable cause. Samsca should not be re-initiated in patients unless the cause for the observed liver injury is definitively established to be unrelated to treatment with Samsca.

CONCLUSIONS

- Loss of sodium along with depletion of volume serves as a signal for the release of AVP.

- As a result of AVP-stimulated retention of water, blood sodium becomes diluted and hyponatremia results.

- VRAs are clearly the most exciting addition to the existing armamentarium for the treatment of hyponatremia.

- Antagonism of V 1 a receptor in vascular smooth muscle cells results in vasodilatation, and antagonism of V 2 receptors in the renal collecting duct results in aquaresis.

- Intravenous Conivaptan, a combined V 1 a/V 2 receptor antagonist, has been approved for the treatment of euvolemic and hypervolemic hyponatremia. Tolvaptan, an oral V 2 receptor antagonist is being evaluated for the treatment of hyponatremia in heart failure. Future indications of vaptans are likely to increase.

References• http://

www.ijabmr.org/article.asp?issn=2229-516X;year=2012;volume=2;issue=2;spage=77;epage=83;aulast=Aditya

• http://www.nejm.org/doi/pdf/10.1056/NEJMoa1205511• http://www.medscape.org/viewarticle/736415_4

Questions