A. Cyclosporine A (CsA, Sandimmune®) [1,2]

1. Cyclic peptide with potent anti-T cell activities
2. Use
   1. Typically in resistant rheumatologic conditions, especially vasculidites
   2. Lower doses are effective in rheumatoid arthritis (RA) but CsA is 3rd-4th line [3]
   3. May be combined safely with methotrexate (MTX) in RA with benefits
   4. Also used in inflammatory myopathies, some systemic sclerosis patients
   5. Effective in severe psoriasis
   6. Used in refractory ulcerative colitis
3. Dosing
   1. Low doses very effective in resistant RA and systemic lupus
   2. Typically 2-4mg/kg/day; aim for levels <200ng/dl
   3. Response within 2 months or increased dose to max 5mg/kg/day
   4. Stop after 3 months if no response or if renal function deteriorates
4. Patients should be vaccinated and followed closely for renal function
5. Side Effects
   1. Concern for GI intolerance, drug interactions, especially Ca channel blockers
   2. Hyperuricemia with gout attacks (often with tophi) are common
   3. Renal insuffiency often occurs but will not generally progress (creatinine rise <25%)
   4. Drug should be stopped or dose lowered if creatinine increases >30-50%
   5. Addition of CsA to PUVA increases squamous cell cancer risk ~7X [5]
6. Contraindications: current / past malignancy, uncontrolled HTN, renal or liver dysfunction
7. Grapefruit juice inhibits CYP3A4 and should be avoided with CsA [52]

B. Tacrolimus (FK506, Prograf®)

1. Binds to a peptide prolyl isomerase/phosphatase
   1. Specifically, binds to cytosolic proteins FKBP-12 and FKBP-25
   2. Does not bind cyclophilin
2. May have slightly greater efficacy in CsA "resistant" rejection episodes
3. Utility mainly in transplantation
   1. Synergistic combination with sirolimus
   2. Often used in liver transplantation
   3. Also effective in combination in islet cell transplants [6]
   4. Used in hand transplantation with basiliximab, permitted >1 year survival [7]
4. Promising in patients with polymyositis and anti-Jo1 or anti-SNP Abs [8]
5. Grapefruit juice inhibits CYP3A4 and should be avoided with tacrolimus [52]

C. Sirolimus (rapamycin, Rapamune®) [9,10]

1. Approved for prevention of acute renal graft rejection with CsA
   1. Used in combination with CsA, permits CsA dose reduction
   2. Superior to azathioprine as add on therapy with CsA
2. Structurally related to tacrolimus, but blocks a distinct regulatory kinase
   1. This kinase is required for signalling through CD28 pathway
   2. Thus, rapamycin prevents T cell activation by blocking second signal
3. Substantially reduced nephrotoxicity compared with CsA and Tacrolimus
4. Combined with rabbit antithymocyte globulin induction, can maintenance monotherapy [11]
5. Main Side Effects
   1. Thrombocytopenia and leukopenia
   2. Hyperlipidemia
   3. Arthralgias and rash commonly occur
6. Dose
   1. Loading dose: 6mg then 2mg/d 4 hours after CsA is taken
   2. Dose as monotherapy in renal transplant is 15mg initially, then 5mg qd with modulated trough levels of 10-15µg/L [11]
   3. Grapefruit juice inhibits CYP3A4 and should be avoided with sirolimus [52]

D. Everolimus [27]

1. Proliferation inhibitor / immunosuppressant similar to sirolimus (Rapamune®)
2. Superior to azathioprine for reducing severity and incidence of cardiac allograft CAD
3. Combined with CsA, also reduced risk of rejection
4. Everolimus + CsA reduced incidence of rejection and CMV infection in kidney allograft

E. Mycophenolate Mofetil (CellCept®) [12]

1. Impairs de novo purine synthesis
   1. Hydrolyzed in vivo to mycophenolic acid which inhibits inosine monophosphate
   2. Mainly inhibits B cell proliferation and antibody (Ab) production
   3. Minimal effects on T lymphocytes
2. Approved for use in reduction in acute renal allograft rejection
3. Possible utility in ANCA-positive vasculitis, IgA nephropathy, RA
4. SLE
   1. Replacing azathioprine in some patients with moderate to severe systemic lupus
   2. Efficacy similar to cyclophosphamide in severe lupus nephritis (used with prednisolone) [13]
   3. Effective and generally well tolerated as maintenance therapy in severe lupus after induction with cyclophsophamide [54]
5. Dose is 0.5-1.5gm po bid
6. Side Effects: diarrhea, leukopenia

F. Intravenous Immunoglobulin (IVIg) [14,15,16]

1. IVIg is prepared from human serum from 100-10,000 donors per course
2. Poorly understood immunomodulating activities
   1. Alteration and blocking Fc receptors on effector cells (particularly macrophages)
   2. May block adherance to endothelial cells and reduce tissue migration
   3. Influencing idiotype-anti-idiotype networks
   4. Transforming Growth Factor ß has been detected in all commercial IVIg's
   5. Attenuation of complement mediated damage
   6. IVIg can increase the rate of IgG catabolism through IgG transport receptor FcRn
   7. Induction of anti-inflammatory cytokines and reduction of inflammatory cytokines
3. Dose is usually 400mg/kg to 2gm/kg per day for 3-5 days (extremely large doses)
4. FDA Approved Indications [14]
   1. Allogeneic bone marrow transplant
   2. Chronic Lymphocytic Leukemia
   3. Idiopathic Thrombocytopenia Purpura (ITP)
   4. Kawasaki Disease
   5. Pediatric HIV
   6. Primary Immunodeficiencies
5. Clearly Established Efficacy
   1. ITP
   2. Guillain-Barre Syndrome (GBS)
   3. Chronic inflammatory demyelinating neuropathy (CIDP)
   4. Multifocal motor neuropathy
   5. Myasthenia gravis (MG; second line)
   6. Dermatomyositis (2nd line)
   7. Kawasaki Disease
   8. Prevention of graft versus host disease in allogeneic transplantation
   9. Stiff-Person (Stiff-Man) Syndrome (2nd line)
   10. Eaton-Lambert Myasthenic Syndrome (2nd line)
   11. Pemphigus - last resort therapy
6. Possible Efficacy
   1. Systemic Lupus Erythematosus - with thrombocytopenia or renal disease
   2. Lupus nephritis - maintenance therapy after cyclophosphamide induction [18]
   3. Primary antiphospholipid syndrome
   4. Vasculitis - particularly ANCA+
   5. Autoimmune uveitis
   6. Multiple Sclerosis (MS)
   7. Toxic Epidermal Necrolysis / Stevens-Johnson Syndrome
   8. Sepsis syndorme - especially septic shock (25% mortality reduction in meta-analysis) [65]
7. Well tolerated overall, but some infrequent side effects
   1. Aseptic Meningitis - severe headache, meningismus, cerebrospinal fluid (CSF) leukocytosis within 48 hours of dosing
   2. Migraine headache
   3. Anaphylactic reactions in some persons with IgA deficiency
   4. Increase in serum viscosity (hyperviscosity syndromes can result)
   5. Mild systemic symptoms during infusions are not unusual including myalgias, back aches
   6. Transmission of viral agents (such as Hepatitis C virus, HCV)
   7. Renal tubular necrosis - extremely rare
   8. Infusion of IVIg increases ESR, can reduce sodium, and can increase antiviral Ab titers
8. A variety of IVIg products are available

G. Tumor Necrosis Factor Alpha (TNFa) Blockade [20,21,22,43]

1. TNFa plays a key role in perpetuation of inflammation and joint destruction in RA
2. Produced by macrophages and lymphocytes
3. TNFa production is stimulated by T lymphocyte (Th1) cytokine production
4. TNFa, IL1, and IL6 play key roles in destruction of joints and bone
5. Indications
   1. Moderate and severe RA
   2. Moderate and severe Crohn's disease; good healing of Crohns' fistulae
   3. Psoriatic arthritis and moderate to severe psoriasis
   4. Ankylosing spondylitis
   5. No benefit of infliximab in polymyalgia rheumatica (PRM) [65] or giant cell arteritis (GCA) [66]
6. Agents
   1. Etanercept
   2. Infliximab
   3. Adalimumab
7. Side Effects
   1. Injection site reactions
   2. Antinuclear antibodies (usually asymptomatic)
   3. Malignancy risk ~3.3X, usually with with higher dose anti-TNFa Abs, mainly non-Hodgkin's lymphomas [4]
   4. Serious infection risk ~2.0X, usually treated with higher dose anti-TNFa Abs [4]
8. Antinuclear Antibodies (ANA)
   1. 5-10% patients on TNFa blockade develop positive ANAs
   2. 5-15% of patients also produce anti-double stranded DNA (dsDNA) Abs
   3. Patients with positive autoantibodies rarely show clinical consequences
   4. Drug induced lupus (DLE) has been reported for both infliximab and etanercept [25]
   5. DLE typically resolves with stopping TNFa blocker ± glucocorticoids
9. Pretreatment Evaluation and Monitoring
   1. Evaluate for latent tuberculosis prior to initiating therapy
   2. Be alert for tuberculosis, histoplasmosis, other infections during treatment
   3. Prior to treatment with TNFa blockers, obtain baseline ANA and dsDNA Abs [25,48]
   4. Case reports of exacerbation or new onset congestive heart failure (rare) [19]

H. Etanercept (Enbrel®) [3,21,26,27,28]

1. Soluble TNFa receptor (p75 dimer with Ig-FcG1)
2. Absorbs and neutralizes TNFa biological activity (complex is cleared)
3. Dose
   1. Adult: 10mg or 25mg sc injections twice weekly (half-life ~5 days)
   2. Children: 0.4mg/kg sc twice weekly for juvenile chronic arthritis [29]
4. Efficacy
   1. Improves moderate and severe RA in 50-70% of patients and JCA
   2. Synergistic in RA when combined with MTX
   3. Clearly slows joint destruction and well tolerated in RA for >18 months
   4. Effective in patients with psoriatic arthritis and severe psoriasis [30]
   5. Significantly reduced psoriasis severity in age 4-17 years with plaque psoriasis [17]
   6. Very effective in severe psoriasis (doses up to 50mg sc twice weekly) [50]
   7. Effective in patients with ankylosing spondylitis [31]
   8. Of no benefit for maintenance therapy in WG when added to standard glucocorticoids and MTX [56]
5. Side Effects
   1. Antinuclear and anti-dsDNA antibodies, anticardiolipin antibodies, rarely symptomatic [48]
   2. Injection site reactions common, but no other major problems
   3. Does not appear to increase risk of tuberculosis [23]
   4. Exacerbation of congestive heart failure (CHF) reported rarely

I. Infliximab (Remicade®) [3,21,26,32]

1. Monoclonal chimeric mouse-human Ab with 10 day half-life (intravenous)
2. Dose 3-10mg/kg IV at 0, 2, 6 weeks, then every 8 weeks
3. Approved severe Crohn's Disease and in severe RA
   1. In Crohn's Disease, responses usually occur at 2 weeks, last ~3 months [24]
   2. Very effective for healing fistulas in Crohn's Disease
   3. ~70% of patients heal >50% and ~55% heal all fistulas [33]
   4. Good maintenance of Crohn's: 5mg/kg at weeks 2 and 6, then 5-10mg/kg q8 weeks [28,53]
   5. Modest to good activity in induction and maintenance in ulcerative colitis [60]
   6. Well tolerated and effective therapy in ~50% of patients with refractory RA who have suboptimal responses to MTX [34]
   7. Effective in moderate to severe plaque psoriasis [35]
   8. In phase 3 study in moderate-severe psoriasis, >75% improvement in up to 80% of patients [59]
   9. Significant disease regression in >50% of ankylosing spondylitis patients [36]
4. No benefit of infliximab in polymyalgia rheumatica (PRM) [65] or giant cell arteritis (GCA) [66]
5. Side Effects
   1. Overall well tolerated with nausea most common (17%)
   2. Immunogenicity to infliximab reduces efficacy and half-life of the agent in Crohn's [37]
   3. Associated with increased risk of local and disseminated tuberculosus [23]
   4. Combined infliximab + immunosuppressive therapy reduces infliximab immunogenicity [37]
   5. Exacerbation of CHF reported rarely
   6. Autoantibodies similar to etanercept [48]

J. Adalimumab (D2E7, Humira®) [21,22]

1. Effective alone or in combination with MTX for moderate to severe RA
2. Convenient dosing: 40mg sc every other week
3. Similar side effects as other TNFa inhibitors
4. Most convenient dosing of all of the agents

K. Anakinra (Kineret®) [5,21]

1. Recombinant non-glycosylated form of natural antagonist to IL1
2. Modest activity in moderate or severe RA
3. May be used alone or in combination with MTX
4. Marked efficacy in NOMID (neonatal onset multisystem inflammatory disease) [62]
5. Side Effects: ruritus, rash, erythema, pain at injection site, neutropenia (8%)
6. Combination with etanercept led to 7% rate or serious infection
7. Dose is 100-150mg daily sc

L. Efalizumab (Anti-CD11a McAb) [51]

1. Binds alpha subunit of LFA1 (CD11a) and blocks interaction with ICAM-1
2. Inhibits T cell trafficking, particularly memory cells
3. May also reduce activation of T lymphocytes
4. About 25% of patients receiving 1-2mg/kg sc efalizumab weekly for 12 weeks had 75% reduction in psoriasis activity and severity
5. Generally well tolerated with some increase in headache, chills, fever, injection site pain

M. Leflunomide (Arava®) [3,20,21,26]

1. Novel isoxazole drug which inhibits pyrimidine synthesis
2. Blocks dihyrdroorate dehydrogenase
3. Immunosuppressive and antiproliferative activities
4. Efficacy in RA [20]
   1. Reduces joint destruction over 24 and 48 week periods
   2. As effective as sulfasalazine and better tolerated [38]
   3. Likely as effective as MTX
   4. May be combined with MTX cautiously [39]
   5. Good benefits but slightly inferior to MTX in polyarticular juvenile RA [57]
5. Metabolized by liver with half life of ~14 days in blood
   1. Much longer half life (3-6 months in the body)
   2. Carcinogenic and teratogenic in animals
   3. If conception desired, must take choletsyramine 8 grams tid for 11 days
   4. If cholestyramine not used, drug takes ~2 years to leave the body
6. Active metabolite blocks CYP2C9
7. Dose is 100mg qd x 3 days (loading), then 20mg/d (or 10mg/d if not well tolerated)
8. Side Effects
   1. Diarrhea is most common
   2. Rash and reversible alopecia also occur
   3. Aminotransferase (liver function) levels increase >3X normal in ~10% of patients
   4. Liver function testing is required (for example, monthly)
   5. Anaphylaxis and leukocytoclastic vasculitis also reported

N. Thalidomide (Thalomid®) [40,41]

1. Activities
   1. Anti-inflammatory and immunomodulatory activities
   2. Stimulates Th1 cytokines: interferon gamma (IFNg), interleukin (IL)-12
   3. Stimulates production of tumor necrosis factor alpha (TNFa) by monocytes
   4. Inhibits angiogenesis
   5. Stimulates CD4+ and CD8+ lymphocytes; increases IL-2R expression
2. Utility
   1. Efficacy in resistant autoimmune cases, especially for aphthous ulcers
   2. Active against severe SLE skin disease
   3. Reduces mucocutaneous lesions in Behcet's [42]
   4. Lepromatous leprosy (ENL reaction)
   5. Graft versus host disease (GVHD)
   6. Has shown efficacy for HIV associated aphthous ulcers, but increased viral load
   7. Responses in ~30% of patients with refractory multiple myeloma [44]
3. Dose
   1. Initiate at 50mg po qd
   2. For erythema nodosum leprosum 100-300mg po qd
   3. For aphthous ulcers, 50-300mg po qd
   4. For multiple myeloma, 50-400mg po qd
   5. Treatment continues until conditions subside
4. Side Effects [55]
   1. Severe teratogenicity
   2. Drowsiness and sedation - most common
   3. Abdominal Pain/Bloating, Constipation - very common
   4. Peripheral Neuropathy - mainly sensory
   5. Orthostatic Hypothension
   6. Dry mouth and/or skin also occur
   7. Thromboembolic Disease
5. Due to high teratogenicity, registration with manufacturer required by physicians
6. Peripheral Neuropathy
   1. Mainly sensory, painful
   2. Occurs in 30-50% of persons with >6 months of use
   3. Appears to be independent of dose but increases with duration of use
   4. Includes "pins and needles", leg cramps, foot pain
   5. EMG/NCS should be done at baseline and every 6 months or 10grams thereafter
   6. Decrease in sensory amplitudes by >40% should prompt discontinuation of drug
7. Thromboembolic Disease [45,55]
   1. Deep vein thrombosis, pulmonary embolism
   2. Incidence with thalidomide alone ~5%
   3. Incidence substantially increased with chemotherapy, particularly doxorubicin
8. Combination with glucocorticoids may also exacerbate side effects

O. Abatacept (CTLA4-Ig, Orencia®) [49,58]

1. Soluble fusion molecule of cytotoxic T clymphocyte antigen 4 with human IgG1 Fc
2. Blocks costimulation of T cells through CD80 and CD86 binding to T lymphocyte CD28
3. Activity in psoriasis and RA
4. CTLA4-Ig 10mg/kg IV over 30 minutes on days 1,15, 30 and then monthly for 6 months
5. Improved responses in RA patients with active disease on MTX
6. Abatacept on days 1,15,29 then monthly x 6 months induced ACR20 responses in 50% of TNFa blocker refractory RA compared with 20% with placebo [58]
7. Abatacept+MTX superior to placebo+MTX in patients on MTX with active RA when given once monthly for 1 year; minimal side effects in combination group [61]
8. No increase in serious infections and minimal adverse events

P. Protein A Column (Prosorba®) Plasmapheresis [46]

1. Protein A is a staphylococcal protein that binds to human immunoglobulin G
2. Approved for use for treatment of resistant RA
3. May be effective in ~30% of patients who fail or are intolerant of MTX
4. Also approved for treatment of immune thrombocytopenia
5. Treatment is weekly for 12 weeks; responses seen after 12 weeks of therapy

Q. Chaperonin (Heat Shock Protein) 10 [63,64]

1. Toll-like receptors (TLR): receptors for "danger" signals linking innate and adaptive immunity
2. TLR are primary source of inflammatory molecules IL-1 and TNFa
3. TLRs are highly expressed in synovial tissue of patients with RA
4. Chaperonin 10 (also called heat shock protein or HSP 10) inhibits TLR expression
5. HSP 10 blocks TLR activation by HSP 60
6. In patients with RA, Chaperonin 10 (HSP 10) protein IV weekly improved RA symptoms [64]

References

1. Langford CA, Klippel JH, Balow JE, et al. 1998. Ann Intern Med. 128(12):1021
2. Langford CA, Klippel JH, Balow JE, et al. 1998. Ann Intern Med. 129(1):49
3. Kremer JM. 2001. Ann Intern Med. 134(8):695
4. Bongartz T, Sutton AJ, Sweeting MJ, et al. 2006. JAMA. 295(19):2275
5. Marcil I and Stern RS. 2001. Lancet. 358(9287):1042
6. Shapiro AMJ, Lakey JRT, Ryan EA, et al. 2000. NEJM. 343(4):230
7. Jones JW, Gruber SA, Barker JH, Breidenbach WC. 2000. NEJM. 343(7):468
8. Oddis CV, Sciurba FC, Elmagd KA, Startzl TE. 1999. Lancet. 353(9165):1762
9. Sirolimus (Rapamycin). 2000. Med Let. 42(1071):13
10. Kahan BD. 2000. Lancet. 356(9225):194
11. Swanson SJ, Hale DA, Mannon RB, et al. 2002. Lancet. 360(9346):1662
12. Myophenolate. 1995. Med Let. 37(958):84
13. Chan TM, Li FK, Tang CSO, et al. 2000. NEJM. 343(16):1156
14. Intravenous Immunoglobulin. 2006. Med Let. 48(1249):101
15. Kazatchkine MD and Kaveri SV. 2001. NEJM. 345(10):747
16. Dalakas MC. 2004. JAMA. 291(19):2367
17. Paller AS, Sigfried EC, Langley RG, et al. 2008. NEJM. 358(3):241
18. Boletis JN, Ionnidis JPA, Boki KA, Moutsopoulos HM. 1999. Lancet. 354(9178):569
19. Kwon HJ, Cote TR, Cuffe MS, et al. 2003. Ann Intern Med. 138(10):807
20. Drugs for Rheumatoid Arthritis. 2000. Med Let. 42(1082):57
21. Olsen NJ and Stein CM. 2004. NEJM. 350(21):2167
22. Adalimumab. 2003. Med Let. 45(1153):25
23. Keane J, Gershon S, Wise RP, et al. 2001. NEJM. 345(15):1098
24. Bell S and Kamm MA. 2000. Lancet. 355(9207):858
25. Shakoor N, Michalska M, Harris CA, Block JA. 2002. Lancet. 359(9306):579
26. Leflunomide and Etanercept. 1998. Med Let. 40(1040):110
27. Weinblatt ME, Kremer JM, Bankjurst AD, et al. 1999. NEJM. 340(4):253
28. Moreland L, Schiff MH, Baumgartner SW, et al. 1999. Ann Intern Med. 130(6):478
29. Lovell DJ, Giannini EH, Reiff A, et al. 2000. NEJM. 342(11):763
30. Mease PJ, Goffe BS, Metz J, et al. 2000. Lancet. 356(9227):385
31. Gorman JD, Sack KE, Davis JC Jr. 2002. NEJM. 346(18):1349
32. Infliximab. 1999. Med Let. 41(1047):19
33. Present DH, Rutgeerts P, Targan S, et al. 1999. NEJM. 340(18):1398
34. Maini R, St Clair EW, Breedveld F, et al. 1999. Lancet. 354(9194):1932
35. Chaudhari U, Romano P, Mulcahy LD, et al. 2001. Lancet. 357(9271):1842
36. Braun J, Brandt J, Listing J, et al. 2002. Lancet. 359(9313):1187
37. Baert F, Noman M, Vermeire S, et al. 2003. NEJM. 348(7):601
38. Smolen JS, Kalden JR, Scott DL, et al. 1999. Lancet. 353(9149):259
39. Kremer JM, Genovese MC, Cannon GW, et al. 2002. Ann Intern Med. 137(9):726
40. Thalidomide. 1998. Med Let. 40(1038):103
41. Calabrese L and Fleischer AB Jr. 2000. Am J Med. 108(6):487
42. Hamuryudan V, Mat C, Saip S, et al. 1998. Ann Intern Med. 128(6):443
43. Scott DL and Kingsley GH. 2006. 355(7):704 (Case Discussion)
44. Singhal S, Mehta J, Desikan R, et al. 1999. NEJM. 341(21):1565
45. Bennett CL, Schumock GT, Desai AA, et al. 2002. Am J Med. 113(7):603
46. Prosorba Columns. 1999. Med Let. 41(1058):69
47. Eisen HJ, Tuzcu EM, Dorent R, et al. 2003. NEJM. 349(9):847
48. Krause I, Valesini G, Scrivo R, Shoenfeld Y. 2003. Am J Med. 115(5):390
49. Kremer JM, Westhovens R, Leon M, et al. 2003. NEJM. 349(20):1907
50. Leonardi CL, Powers JL, Matheson RT, et al. 2003. NEJM. 349(21):2014
51. Lebwohl M, Tyring SK, Hamilton TK, et al. 2003. NEJM. 349(21):2004
52. Drug Interactions with Grapefruit Juice. 2004. Med Let. 46(1773):2
53. Sands BE, Anderson FH, Bernstein CN, et al. 2004. NEJM. 350(9):876
54. Contreras G, Pardo V, Leclercq B, et al. 2004. NEJM. 350(10):971
55. Dimopoulos MA and Eleutherakis-Papaiakovou V. 2004. Am J Med. 117(7):508
56. Wegener's Granulomatosis Etanercept Trial Research Group. 2005. NEJM. 352(4):351
57. Silverman E, Mouy R, Spiegel L, et al. 2005. NEJM. 352(16):1655
58. Genovese MC, Becker JC, Schiff M, et al. 2005. NEJM. 353(11):1114
59. Reich K, Nestle FO, Papp K, et al. 2005. Lancet. 366(9494):1367
60. Rutgeerts P, Sandborn WJ, Feagan BG, et al. 2005. NEJM. 353(23):2462
61. Kremer JM, Genant HK, Moreland LW, et al. 2006. Ann Intern Med. 144(12):865
62. Golbach-Mansky R, dailey NJ, Canna SW, et al. 2006. NEJM. 355(6):581
63. Emery P and Ponchel F. 2006. Lancet. 368(9538):821
64. Vanags D, Williams B, Johnson B, et al. 2006. Lancet. 368(9538):855
65. Salvarani C, Macchioni P, Manzini C, et al. 2007. Ann Intern Med. 146(9):631
66. Hoffman GS, Cid MC, Rendt-Zagar KE et al. 2007. Ann Intern Med. 146(9):621