USO09556260B2

(12) United States Patent (10) Patent No.: US 9,556.260 B2 Frey, II et al. (45) Date of Patent: Jan. 31, 2017

(54) TREATMENT OF CENTRAL NERVOUS 2004/0101909 A1 5/2004 Lemieux et al. SYSTEM DSORDERS BY INTRANASAL 3988) A. 1858. 1 rey, 11 et al. ApMINISTRATION OF IMMUNOGLOBULIN 2011 O151393 A1 6/2011 Frey, II et al.

(71) Applicants: Baxalta Incorporated, Bannockburn, FOREIGN PATENT DOCUMENTS IL (US); Baxalta GmbH, Glattpark WO WO 96,228O2 A1 8/1996 (Opfikon) (CH) WO WO O1 (60420 A1 8, 2001 WO WO 03/028668 A2 4/2003 (72) Inventors: yO O ES E. Bear Lake, WO WO 2006/0913322007/0956.16 A2 8, 20072006 (US); Leah Ranae Bresin WO WO 2007/106617 A2 9, 2007 Hanson, Vadnais Heights, MN (US); WO WO 2009/058957 A2 5, 2009 Sharon Pokropinski, Schaumburg, IL WO WO 2009, 111240 A1 9, 2009 (US); Francisco M. Rausa, III, Vernon WO WO 2011/095.543 A1 8, 2011 Hills, IL (US) OTHER PUBLICATIONS (73) Assignees: Baxalta Incorporated, Bannockburn, IL(Opfikon) (US); Balxalta (CH) GmbH, Glattpark particularly,R sia Table Milityac InStration R for Rsp. p. 383-393 Bohmwald et al., Rev Med Virol. 24:407-419, Oct. 2014 (evidence (*) Notice: Subject to any disclaimer, the term of this hasE"ts with CNS conditions such as fever, and patent is extended or adjusted under 35 Cattepoel et al. PLoS One, vol. 6, Issue 4: e18296, Apr. 2011.* U.S.C. 154(b) by 0 days. Chu LW. Hong Kong Med J., 18(3) Jun. 2012.* Kelly et al., J Appl Physiol. 89:323-337, 2000.* (21) Appl. No.: 14/189,981 Magga et al. J Neuroinflam., 7.90, 2010.* Widiaprada et al. J Neuroschem., 122:321-332, 2012.* (22) Filed:1-1. Feb. 25, 2014 2013Kolobov abstract et al. only.*Bulletin of Exp. Biol. Med., 154(4): 425-427, Feb. O O Xiao et al., Program No. 853.14. 2012 Neuroscience Meeting (65) US t A. New Orleans, LA: Society for Neuroscience, 2012. lug. ZS, Xiao et al., Journal of Alzheimer's Disease, vol. 35. No. 4, pp. T77-788, Ju124, 2013. Related U.S. Application Data Chauhan etal Alzhiemer's & , vol. 8,sy issue S4 poster p. (60) Provisional application No. 61/769,673, filed on Feb. 1-261, Jul. 14-19, 2012. 26,s 2013. pprovisional applicationpp No. 61/862.814.s s DemyelinationAkassoglou, K. Induced et al., byOligodendrocyte. Local TNFp55TNF Apoptosis Receptor and Signaling Primary filed on Aug. 6, 2013. in the Central of Transgenic Mice.” American Journal of Pathology, Sep. 1998, vol. 153, No. 3, pp. 801-813. (51) Int. Cl. Athwal, G.S., “The Emergence of Antibody Fragments and Deriva C07K 6/06 (2006.01) tives.” Innovations in Pharmaceutical Technology, Jul. 2009, pp. C07K 6/8 2006.O1 46-48. A6 IK 9/12 30: 8: Awad, A. et al., “Idiopathic Transverse and Neuromyelitis Optica:p Clinical Profiles, Pathophysiolophy gy and Therapeuticp A6IP 25/28 (2006.01) Choices.” Current Neuropharmacology, 2011, vol. 9, pp. 417-428. A6 IK3I/98 (2006.01) Bohmwald, K. et al., “Central nervous system alterations caused by A6 IK 3/472 (2006.01) infection with the human respiratory syncytial virus,” Rev. Med. A6 IK 3/40 (2006.01) Virol., 2014, 13 pages A6 IK 9/00 (2006.01) Bolli, Ret al., -Proline reduces IgG dimer content and enhances A61 K 39/00 (2006.01) tologicals,by 5. , Vol.yers 58, pp.in globulinF , (IVIG) solutions, (52) 3. (2006.01) (Continued) CPC ...... C07K 16/18 (2013.01); A61K 9/0043 Primary Examiner – Kimberly A. Ballard (2013.01); A61K 31/198 (2013.01); A61 K Assistant Examiner — Stacey N MacFarlane 31/401 (2013.01); A61K 31/4172 (2013.01); (74). Attorney, Agent, or Firm — Morgan, Lewis & C07K 16/06 (2013.01); A61 K 9/08 (2013.01); Bockius LLP A61K 2039/505 (2013.01); A6 IK 2039/543 (57) ABSTRACT (2013.01). C07K 231 7/55 (2013.01) The present invention provides, among other aspects, meth (58) Field of Classification Search ods and compositions for treating a central nervous system None (CNS) disorder by delivering a therapeutically effective See application file for complete search history. amount of a composition of pooled human immunoglobulin G (IgG)9. to the brain via intranasal administration of the (56) References Cited composition directly to the olfactory epithelium of the nasal cavity. In particular, methods and compositions for treating U.S. PATENT DOCUMENTS Alzheimer's disease are provided. 5,624,898 A 4/1997 Frey, II 31 Claims, 28 Drawing Sheets 6,715.485 B1 4/2004 Djupesland (9 of 28 Drawing Sheet(s) Filed in Color) US 9,556.260 B2 Page 2

(56) References Cited Orbach, H. et al., “Intravenous Immunoglobulin, Adverse Effects and Safe Administration.” Clinical Reviews in Allergy and Immu nology, 2005, vol. 29, pp. 173-184. OTHER PUBLICATIONS Pardridge, W.M., “Drug and Gene Targeting to the Brain with Molecular Trojan Horses.” Nature Reviews, Feb. 2002, vol. 1. pp. Cattepoel, S. et al., “Chronic Intranasal Treatment with an Anti 131-139. A?o ScFv Antibody Ameliorates Amyloid Pathology in a Trans Pardridge, W.M.. “Blood-Brain Barrier Drug Targeting: The Future genic Mouse Model of Alzheimer's Disease.” PLoS One, Apr. 2011, Development of Brain Drug Development.” Molecular Interven vol. 6, No. 4, pp. 1-13. tions, Mar. 2003, vol. 3, No. 2, pp. 90-105. Dodel, R.C. et al., “Intravenous immunoglobulins containing anti Patrias, L.M. et al., “Specific antibodies to soluble alpha-synuclein bodies against B-amyloid for the treatment of Alzheimer's disease.” conformation in intravenous immunoglobulin preparations.” Clini J Neurol Neurosurg Psychiatry, 2004, vol. 75, pp. 1472-1474. cal and Experimental Immunology, 2010, vol. 161, pp. 527-535. Dodel, R. et al., “Intravenous immunoglobulin for treatment of Perl, D.P., “Neuropathology of Alzheimer's Disease and Related mild-to-moderate Alzheimer's disease: a phase 2, randomised, Disorders.” Dementia, Nov. 2000, vol. 18, No. 4, pp. 847-864. double-blind, placebo-controlled, dose-finding trial.” Lancet Perlmutter, S.J. et al., “Therapeutic plasma exchange and intrave Neurol, 2013, vol. 12, pp. 233-243. nous immunoglobulin for obsessive-compulsive disorder and tic Elovaara, I. et al., “Intravenous Immunoglobulins Area. Therapeutic disorders in childhood.” The Lancet, Oct. 2, 1999, vol. 354, pp. Option in the Treatment of Relapse.” Clinical 1153-1158. Neuropharmacology, Mar/Apr. 2011, vol. 34, No. 2, pp. 84-89. Pohl, D. et al., “Treatment of Acute Disseminated Encephalomy Ertekin-Taner, N., “Genetics of Alzheimer's Disease: A Centennial elitis.” Current Treatment Options in , 2012, vol. 14, pp. Review.” Neurol Clin., Aug. 2007, vol. 25, No. 3, 43 pages. 264-275. Fillit, H. et al., “IV immunoglobulin is associated with a reduced Puli, L. et al., “Effects of human intravenous immunoglobulin on risk of Alzheimer disease and related disorders.” Neurology, 2009, amyloid pathology and neuroinflammation in a mouse model of vol. 3, pp. 180-185. Alzheimer's disease,” Journal of Neuroinflammation, 2012, vol. 9, Fu, H.J. et al., “Amyloid-f Immunotherapy for Alzheimer's Dis No. 105, pp. 1-19. ease.” CNS Neurol Disord Drug Targets, Apr. 2010, vol. 9, No. 2, Relkin, N.R. et al., "18-Month study of intravenous immunoglobu pp. 197-206. lin for treatment of mild Alzheimer's disease.” Neurobiology of Haley, M. et al., “The Role for Intravenous Immunoglobulin in the Aging, 2009, vol. 30, pp. 1728-1736. Treatment of West Nile Virus .” Clinical Infectious Smith, L.M. et al., “Effects of intravenous immunoglobulin on alpha Disease, Sep. 15, 2003, vol. 37, pp. e-88-90. Synuclein aggregation and neurotoxicity.” International Harmsen, M.M. et al., “Properties, production, and applications of Immunopharmacology, 2012, vol. 14, pp. 550-557. camelid single-domain antibody fragments.' Appl Microbiol Snider, L.A. et al., “Childhood-Onset Obsessive-Compulsive Dis Biotechnol, 2007, vol. 77, pp. 13-22. order and Tic Disorders: Case Report and Literature Report.” Imbach, P. et al., “High-Dose Intravenous Gammaglobulin for Journal of Child and Adolescent Psychopharmacology, 2003, vol. Idiopathic Thrombocytopenic Purpura in Childhood.” The Lancet, 13, Suppl 1, pp. S81-S88. Jun. 6, 1981, pp. 1228-1231. Stangel, M.. “New advances in the treatment of neurological International Search Report mailed Jun. 17, 2014, for International diseases using high dose intravenous immunoglobulins.” Therapeu Patent Application No. PCT/US2014/018426, 5 pages. tic Advances in Neurological Disorders, 2008, vol. 1, No. 2, pp. Johnson, N.J. et al., “Trigeminal pathways deliver a low molecular 115-124. weight drug from the nose to the brain and orofacial structures.” Mol Stevenson, B.R. et al., “The epithelial tight junction: Structure, Pharm., Jun. 7, 2010, vol. 7, No. 3, pp. 884-893. function and preliminary biochemical characterization.” Molecular Kraus, D. et al., “Schilder's disease: Non-invasive diagnosis and and Cellular Biochemistry, 1988, vol. 83, pp. 129-145. Successful treatment with human immunoglobulins.” Official Jour Stiehm, E.R., "Lessons From Kawasaki Disease: All Brands of nal of the European Paediatric Neurology Society, 2012, vol. 16, IVIG Are Not Equal.” The Journal of Pediatrics, Jan. 2006, pp. 206-207. Editorials, pp. 6-8. Magga, J. et al., “Human intravenous immunoglobulin provides Vo, A.A. et al., “Safety and Adverse Events Profiles of Intravenous protection against Afs toxicity by multiple mechanisms in a mouse Gammaglobulin Products Used for Immunomodulation: A Single model of Alzheimer's disease,” Journal of Neuroinflammation, Center Experience.” Clin J Am Soc Nephrol. 2006, vol. 1, pp. 2010, vol. 7, No. 90, pp. 1-15. 844-852. Mirra, S.S. et al., “Making the Diagnosis of Alzheimer's Disease. A Weltzin, R. et al., “Intranasal Antibody Prophylaxis for Protection Primer for Practicing Pathologists.” Diagnosing Alzheimer's Dis against Viral Disease.” Clinical Microbiology Reviews, Jul. 1999, ease, Feb. 1993, vol. 117, No. 2, pp. 132-144. vol. 12, No. 3, pp. 383-393. Orange, J.S. et al., “Use of intravenous immunoglobulin in human Xiao, C. et al., "Brain Transit and Ameliorative Effects of disease: A review of evidence by members of the Primary Immu Intranasally Delivered Anti-Amyloid-f Oligomer Antibody in nodeficiency Committee of the American Academy of Allergy, 5XFAD Mice,” JAlzheimers Dis., 2013, vol. 35, No. 4, pp. 777-778. Asthma and Immunology,” J Allergy Clin Immunol. 2006, vol. 117. pp. S525-S553. * cited by examiner U.S. Patent Jan. 31, 2017 Sheet 1 of 28 US 9,556.260 B2

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i US 9,556.260 B2 1. 2 TREATMENT OF CENTRAL NERVOUS formed largely by deposition of a small amyloid-beta (AB) SYSTEM DSORDERS BY INTRANASAL peptide derived from the amyloid precursor protein (APP). ADMINISTRATION OF IMMUNOGLOBULIN To date, the U.S. Food and Drug Administration (FDA) G has approved two types of medications for the management of Alzheimer's disease: cholinesterase inhibitors, including CROSS REFERENCES TO APPLICATIONS donepezil (e.g., ARICEPTR), rivastigmine (e.g., EXELONR), galantamine (e.g., RAZADYNE(R), and This application claims priority to U.S. Provisional Patent tacrine (e.g., COGNEXR); and the NMDA-type glutamate Application Ser. Nos. 61/769,673 filed Feb. 26, 2013, and receptor inhibitor memantine (marketed under a number of 61/862,814 filed Aug. 6, 2013, the disclosures of which are 10 different brands). Although a cure for Alzheimer's disease hereby incorporated herein by reference in their entireties has not been identified, these therapies serve to alleviate for all purposes. cognitive symptoms such as memory loss, confusion, and loss of critical thinking abilities in Subjects diagnosed with BACKGROUND OF THE INVENTION age-related dementia (e.g., Alzheimer's disease). In all, it is 15 estimated that healthcare spending on Alzheimer's disease The central nervous system (CNS) is the processing and related age-related in 2012 will be $200 center for the nervous system. CNS disorders can affect the billion in the United States alone (Factsheet, Alzheimer's brain, the , and nerve endings, resulting in Association, March 2012). neurological and/or psychiatric disorders. CNS disorders In addition to these approved therapies, several studies can be caused by genetic inheritance, trauma, infection, 20 have suggested that pooled intravenous immunoglobulin autoimmune disorders, structural defects, tumors, and (IVIG) is effective in slowing the progression of symptoms . Certain CNS disorders are characterized as neuro in Alzheimer's patients (Dodel R C et al., J Neurol Neuro degenerative disease, many of which are inherited genetic surg Psychiatry, Oct: 75(10): 1472-4 (2004); Magga J. et al., diseases. Examples of neurodegenerative diseases include J Neuroinflammation, December 7; 7:90 (1997); Relkin NR Huntington's disease, ALS, hereditary spastic hemiplegia, 25 et al., Neurobiol Aging, 30(11):1728-36 (2008); Puli L. et al., primary lateral sclerosis, , Kenne J Neuroinflammation May 29; 9:105 (2012)). dy's disease, Alzheimer's disease, a polyglutamine repeat Immune globulin products from human plasma were first disease, or Parkinson's disease. Treatment of CNS disorders, used in 1952 to treat immune deficiency. Initially, intramus e.g., genetic diseases of the brain such as Parkinson's cular or Subcutaneous administration of immunoglobulin disease, Huntington's disease, and Alzheimer's disease, 30 isotype G (IgG) isolated from plasma were the methods of remain an ongoing problem. choice. However, IgG products that could be administered Alzheimer's disease is a common form of age-related intravenously, referred to as intravenous immunoglobulin dementia that causes gradual loss of cognitive function, (IVIG), were later developed to allow for the administration including memory and critical thinking abilities. Alzheim of larger amounts of IgG necessary for effective treatment of er's disease is diagnosed clinically by through a finding of 35 various diseases. Usually, IVIG contains the pooled immu progressive memory loss and decrease in cognitive abilities. noglobulin G (IgG) immunoglobulins from the plasma of However, confirmation of Alzheimer's disease does not multiple donors, e.g., more than a hundred or more than a occur until after death. thousand blood donors. These purified IgG products are Alzheimer's disease is becoming more prevalent in devel primarily used in treating three main categories of medical oped nations, where an increase in the population of elder 40 conditions: (1) immune deficiencies: X-linked agamma persons has occurred due in part to improved healthcare. globulinemia, hypogammaglobulinemia (primary immune While less than 1% of the population under the age of 60 is deficiencies), and acquired compromised immunity condi affected by Alzheimer's, it is estimated that 25% to 33% of tions (secondary immune deficiencies), featuring low anti persons develop some form of Alzheimer's by the age of 85. body levels; (2) inflammatory and autoimmune diseases; and As of 2012, 5.4 million Americans were diagnosed with 45 (3) acute infections. Alzheimer's. As life expectancy continues to increase Specifically, many people with primary immunodefi worldwide, the prevalence of Alzheimer's and other age ciency disorders lack antibodies needed to resist infection. related dementia should continue to grow as well. In certain cases these deficiencies can be supplemented by Alzheimer's disease is typically classified as either “early the infusion of purified IgG, commonly through intravenous onset,” referring to cases that begin to manifest at between 50 administration (i.e., IVIG therapy). Several primary immu 30 and 60 years of age in affected individuals, and the more nodeficiency disorders are commonly treated in the fashion, common “late onset Alzheimer's, in which symptoms first including X-linked agammaglobulinemia (XLA), Common become apparent after the age of 60. Although only about Variable Immunodeficiency (CVID), Hyper-IgM Syndrome 10% of all Alzheimer's cases are familial, early onset (HIM), Severe Combined Immunodeficiency (SCID), and Alzheimer's disease has been linked to mutations in the 55 some IgG subclass deficiencies (Blaese and Winkelstein, J. amyloid precursor protein (app), presenilin 1 (psen1), and Patient & Family Handbook for Primary Immunodeficiency presenilin 2 (psen2) genes, while late onset Alzheimer's Diseases. Towson, MD: Immune Deficiency Foundation; disease has been linked to mutations in the apolipoprotein E 2007). (apoE) gene (Ertekin-Taner N., Neurol Clin., 25:611-667 While IgG treatment can be very effective for managing (2007)). 60 primary immunodeficiency disorders, this therapy is only a Histopathologically, this neurodegenerative disease is temporary replacement for antibodies that are not being characterized by the formation of amyloid plaques, neuro produced in the body, rather than a cure for the disease. fibrillary tangles, amyloid angiopathy, and granolovacuolar Accordingly, patients depend upon repeated doses of IgG degeneration in the cerebral cortex (Mirra et al., Arch Pathol therapy, typically about once a month for life. This therapy Lab Med., 117:132-144 (1993); Perl D. P. Neurol Clin., 65 places a great demand on the continued production of IgG 18:847-864 (2000)). The characteristic amyloid plaques, compositions. However, unlike other biologics that are pro used to confirm Alzheimer's disease post-mortem, are duced via in vitro expression of recombinant DNA vectors, US 9,556.260 B2 3 4 IgG is fractionated from human blood and plasma donations. Current IgG manufactures typically rely on either a Cohn Thus, the level of commercially available IgG is limited by method 6 Fraction II+III precipitate or a Kistler-Nitschmann the available Supply of blood and plasma donations. precipitate A as the starting material for downstream pro Several factors drive the demand for IgG, including the cessing. Both fractions are formed by a two step process in acceptance of IgG treatments, the identification of additional 5 which proteins such as fibrinogen and Factor XIII are indications for which IgG therapy is effective, and increas removed by an initial precipitation step (Fraction I precipi ing patient diagnosis and IgG prescription. Notably, the tation) performed at high pH (7.2) with low ethanol con global demand for IgG more than quadrupled between 1990 centration (8-10% V/v), followed by a second precipitation and 2009, and continues to increase at an annual rate step in which IgG is precipitated from the Fraction I super between about 7% and 10% (Robert P., Pharmaceutical 10 natant at pH 6.8 with 20-25% (v/v) ethanol (Fraction II+III) Policy and Law, 11 (2009) 359-367). For example, the or at pH 5.85 with 19% ethanol (v/v) ethanol (precipitate A), Australian National Blood Authority reported that the while albumin and a significant portion of A1PI remain in demand for IgG in Australia grew by 11.1% for the 2010 the Supernatant. 2011 fiscal year (National Blood Authority Australia Annual These methods, while laying the foundation for an entire Report 2010-2011). 15 industry of plasma derived blood proteins, were unable to It has been reported that in 2007, 26.5 million liters of provide IgG preparations having Sufficiently high purity for plasma were fractionated, generating 75.2 metric tons of the chronic treatment of several immune-related diseases, IgG, with an average production yield of 2.8 grams per liter including Kawasaki Syndrome, immune thrombocytopenic (Robert P. Supra). This same report estimated that global purpura, and primary immune deficiencies, without an IgG yields are expected to increase to about 3.43 grams per undue occurrence of serious side effects. As such, additional liter by 2012. However, due to the continued growth in methodologies employing various techniques, such as ion global demand for IgG, projected at between about 7% and exchange chromatography, were developed to provide 14% annually between now and 2015, further improvement higher purity IgG formulations. Hoppe et al. (Munch Med of the overall IgG yield will be needed to meet global Wochenschr 1967 (34): 1749-1752), Falksveden (Swedish demand. One of the factors that may drive increased demand 25 Patent No. 348942), and Falksveden and Lundblad (Meth for pooled human immunoglobulins (e.g., IVIG) over the ods of Plasma Protein Fractionation 1980) were among the next decade is whether or not IgG is approved for the first to employ ion exchange chromatography for this pur treatment of Alzheimer's disease. It is estimated that if these pose. treatments are approved by major regulatory agencies, an It is common practice to administer IgG by intravenous additional 5% increase in demand for IVIG will be seen 30 (IV) injection (Imbach et al., Lancet 1 (8232): 1228-31 (Robert P. Supra). (1981)). Intravenous IgG (IVIG) may be administered alone Due in part to the increasing global demand and fluctua or in combination with other compositions. IVIG is often tions in the available Supply of immunoglobulin products, administered over a 2 to 5 hour period, once a day for 2 to several countries, including Australia and England, have 7 days, with follow-up doses every 10 to 21 days or every implemented demand management programs to protect Sup 35 3 to 4 weeks. Such an administration regime is time con plies of these products for the highest demand patients Suming and inconvenient for many patients. This inconve during times of product shortages. Thus, the development of nience may be aggravated in the case of Alzheimer's methodologies that reduce the amount of pooled immuno patients, who may have difficulty sitting quietly during the globulin G needed to treat various indications will be critical infusion period, and may have to rely on their caregiver to as the increase in demand for pooled immunoglobulin 40 bring them to an infusion center or Supervise their infusion. begins to outpace the increase in global manufacturing Systemic IVIG administration may cause adverse side output. effects. For example, IVIG may cause backache, , Pooled human immunoglobulin G (IgG) is manufactured , or muscle pain, general feeling of discom according to different processes depending upon the specific fort, leg cramps, rash, pain at the injection site, hives, manufacturer. However, the origin of most manufacturing 45 dizziness, unusual fatigue or tiredness or weakness, chills, processes is found in the fourth installment of a series of fever, Sweating, increased heart rate, increased blood pres seminal papers published on the preparation and properties Sure, cough, redness of the face, upset stomach, upper of serum and plasma proteins, Cohn et al. (J. Am. Chem. abdominal pain, and vomiting. Immediate adverse effects Soc., 1946, 68(3): 459-475). This paper first described a post-IVIG administration which have been observed include method for the alcohol fractionation of plasma proteins 50 headache, flushing, malaise, chest tightness, fever, chills, (method 6), which allows for the isolation of a fraction myalgia, fatigue, dyspnea, back pain, nausea, Vomiting, enriched in IgG from human plasma. diarrhea, changes, tachycardia, and anaphy The Cohn procedures were initially developed to obtain lactic reactions. Orbach et al., Clin. Rev. Allergy Immunol., albumin at relatively high (95%) purity by fractional pre 29(3): 173-84 (2005). cipitation with alcohol. However, it was realized by Oncley 55 Furthermore, the adverse side effects may vary based on et al. (J. Am. Chem. Soc., 1949, 71(2): 541-550), Deutschet the IVIG manufacturer. Most manufactures preparations al. (J. Biol. Chem., 1946, 164, 109-118), and Kistler and contain between 90% and 99% purified IgG in combination Nitschmann (Vox Sang., 1962, 7, 414-424), that particular with stabilizers and liquid(s) for reconstitution. Orange et al. protein precipitates (Fraction II and Fraction II+III) from the 2006 (J. Allergy Clin. Immunol. 117(4 Suppl.): S525); Voet Cohn method could be used as a starting material for the 60 al. 2006 (Clin. J. Am. Soc. Nephrol. 1 (4): 844: Stiehm et al. purification of highly enriched immunoglobulin composi 2006 (J. Pediatr. 148(1): 6). For example, some manufac tions. In order to achieve the higher purity and safety turers use maltose as a stabilizer while others use Sucrose or required for the intravenous administration of IgG compo amino acids. sitions, several purification and polishing steps (e.g. adsorp The sodium and Sugar content in WIG, along with varying tion in general or all different chromatographic techniques, 65 amounts of IgA and additional chemicals used in the IVIG Cross-flow-filtration, etc.) have been added to IgG manu production can affect the tolerability and efficacy of the facturing processes after the alcohol fractionation steps. brand of IVIG in patients. Specifically, older patients often US 9,556.260 B2 5 6 suffer from co-morbid conditions that increase the risk of molecules, such as intact antibodies, has not yet been IVIG adverse side effects. For example, subjects with renal demonstrated. The difficulty in transporting larger proteins is disorders, vascular disorders, or also have a height believed to be due to the limited permeability of tight ened risk of renal insufficiency and thrombotic events fol junctions present in the olfactory epithelia, which likely lowing IVIG administration because IVIG compositions are 5 excludes globular molecules having a hydrodynamic radius commonly hyper-viscous and contain high concentrations of of more than 3.6 A (Stevenson B R, et al., Mol Cell Sugar and salt. Biochem., 1988 October: 83(2):129-45). IVIG also carries the risk of catheter-related infection, U.S. Pat. No. 5,624,898 to Frey describes compositions i.e., an infection where the catheter or needle enters a and methods for transporting neurologic agents, which pro subjects or skin. Examples of catheter-related infection 10 mote nerve cell growth and Survival or augment the activity are tenderness, warmth, irritation, drainage, redness, Swell of functioning cells, to the brain by means of the olfactory ing, and pain at the catheter site. Accordingly, alternate neural pathway. The neurological agents of the 898 patent modes of administration would be beneficial from the stand are transported to the brain by means of the nervous system, point of time, convenience, and adverse side effects. rather than the , so that potentially thera In addition to adverse side effects of systemic adminis 15 peutic agents that are unable to cross the blood-brain barrier tration of IVIG, penetration of IVIG across the blood-brain may be delivered to damaged neurons in the brain. The barrier has been shown to be unpredictable and intraven compositions described in the 898 patent include a neuro tricular or intrathecal IgG may be necessary. For example, logic agent in combination with a pharmaceutical carrier Haley et al. administered IVIG in the treatment of meningeal and/or additive which promote the transfer of the agent caused by West Nile virus encephalitis. Haley within the olfactory system. The 898 patent does not teach et al. found that penetration of IVIG was unpredictable and intranasal administration of pooled human immunoglobu posited that intrathecal or intraventricular administration lins. may be required. Haley et al. 2003 (Clin. Inf. Diseases 37: PCT publications WO 2006/091332 and WO 2009/ e88-90). 058957, both by Bentz et al., describe methods for the It is difficult to target the CNS with IV administration 25 delivery of therapeutic polypeptides to the brain by fusing therapeutic compositions because of the blood-brain barrier the polypeptide to an antibody or antibody fragment and (BBB). The BBB provides an efficient barrier, preventing administering the resulting fusion protein intranasally. and/or limiting access to the CNS of therapeutic composi Although the examples of the 332 and 957 PCT publica tions administered intravenously into the peripheral circu tions Suggest that Fc-fusion “mimetibodies' may be admin lation. Specifically, the BBB prevents diffusion of most 30 istered intranasally, neither publication demonstrates deliv therapeutic compositions, especially polar compositions, ery of larger, intact antibodies. In fact, the 957 PCT into the brain from the circulating blood. publication, published three years after the 332 PCT pub At least three methods for increasing the passage of lication, states that “in published delivery studies to date, molecules through the BBB have been developed. First, intranasal delivery efficiency to the CNS has been very low lipophilic compounds Such as lipid-soluble drugs and polar 35 and the delivery of large globular macromolecules, such as drugs encased in a lipid membrane have been developed. antibodies and their fragments, has not been demonstrated.” Lipophilic compounds with a molecular weight of less than The 957 PCT publication purports to solve this problem 600 Da can diffuse through the BBB. Second, therapeutic through the use of a permeability enhancer (e.g., membrane compounds can be bound to transporter molecules which fluidizers, tight junction modulators, and medium chain can cross the BBB through a saturable transporter system. 40 length fatty acids and salts and esters thereof, as described Examples of Saturable transporter molecules are transferrin, below), which enhances intranasal administration to the insulin, IGF-1, and leptin. Third, therapeutic compounds can central nervous system. Neither PCT publication teaches cross the BBB by binding the therapeutic compounds to intranasal administration of pooled human immunoglobu polycationic molecules such as positively-charged proteins lins. that preferentially bind to the negatively-charged endothelial 45 PCT publication WO 2003/028668 by Barstow et al., surface of the BBB. Patridge et al. 2003 (Mol. Interv. 3(2): describes the treatment of immune-mediated diseases by 90-105); Patridge et al. 2002 (Nature Reviews-Drug Dis alimentary administration (i.e., administration to the diges covery 1:131-139). However, each of the above-described tive tract) of pooled immunoglobulins. Although the 668 approaches for increasing the delivery of therapeutics PCT publication discloses nasal administration of a compo through BBB to gain access to the CNS are limited. One 50 sition of pooled immunoglobulins, it is in the context of such limitation is that the above-described approaches rely delivering the composition to the digestive tract. The 668 on systemic delivery systems, e.g., administration directly or PCT publication does not teach the delivery of pooled indirectly to the blood stream, which results in non-specific human immunoglobulins to the brain via intranasal admin delivery of the therapeutic agent to other parts in the body, istration. increasing the chance of adverse side effects. 55 PCT publication WO 2001/60420 by Adjei et al., Intranasal administration of therapeutics has become an describes aerosol formulations of therapeutic polypeptides, increasingly explored method for delivering therapeutic including immunoglobulins, for pulmonary delivery. These agents to the brain because it circumvents the BBB and is a aerosolizable compositions are formulated Such that after localized, non-invasive method for delivery. Furthermore, oral or nasal inhalation, the therapeutic agent is effectively intranasal administration offers the advantages, over more 60 delivered to the patient’s lungs. The 420 PCT publication traditional methods of delivery (e.g., intravenous, Subcuta does not teach the delivery of therapeutic agents to the brain neous, oral transmucosal, oral or rectal administration), of via intranasal administration. being simple to administer, providing rapid onset of action, Accordingly, there is a need in the art for methods of and avoiding first-pass metabolism. Unfortunately, intrana treating central nervous system disorders, such as Alzheim sal administration has only been shown effective for the 65 er's disease, using pooled human immunoglobulin G that transport of Small molecules, and to a certain extent Smaller provide specific targeting to the CNS (e.g., administration Fc fusion proteins, to the brain. The delivery of larger primarily to the brain), reduce systemic distribution of the US 9,556.260 B2 7 8 pooled immunoglobulins, and lower the therapeutically or paroxysmal disorder of the central nervous system, a effected dose needed for administration. paralytic syndrome of the central nervous system, a nerve, nerve root, or plexus disorder of the central nervous system, BRIEF SUMMARY OF INVENTION an organic mental disorder, a mental or behavioral disorder caused by psychoactive Substance use, a schizophrenia, The present disclosure provides solutions to these and Schizotypal, or delusional disorder, a mood (affective) dis other problems by providing methods and compositions for order, neurotic, stress-related, or somatoform disorder, a the treatment of central nervous system disorders via intra behavioral syndrome, an adult personality or behavior dis nasal administration of pooled human immunoglobulin G. order, a psychological development disorder, and a child Advantageously, intranasal administration provides directed 10 delivery of pooled IgG to the brain, bypassing the require onset behavioral or emotional disorder. ment that it pass through the blood brain barrier (BBB). As In one embodiment of the methods described above, the shown herein, intranasal administration allows the delivery CNS disorder is selected from the group consisting of of intact IgG to the brain. This results in greater efficiency Alzheimer's disease, Parkinson's disease, multiple Sclerosis, for the treatment and reduces the necessary IgG dose that 15 amyotrophic lateral sclerosis (ALS), Huntington's disease, must be administered to achieve the desired effect. As cerebral palsy, bipolar disorder, schizophrenia, and Pediatric pooled human IgG is isolated from donated human plasma, acute-onset neuropyschiatric syndrome (PANS). pooled IgG is a limited resource. The reduction in the In one embodiment of the methods described above, the effective dose of IgG provided by the present disclosure CNS disorder is selected from the group consisting of effectively increases the therapeutic potential provided by Alzheimer's disease, Parkinson's disease, multiple Sclerosis, the world's supply of pooled human IgG. Furthermore, as Pediatric Autoimmune Neuropsychiatric Disorders Associ demonstrated herein, intranasal administration of IgG nearly ated with Streptococcal infections (PANDAS), and Pediatric eliminates the systemic exposure caused by intravenous acute-onset neuropyschiatric syndrome (PANS). administration, improving the overall safety profile of the In one embodiment of the methods described above, the treatment. Finally, it was Surprisingly found that IgG is 25 CNS disorder is selected from the group consisting of efficiently transported to the brain when intranasally admin Alzheimer's disease, multiple Sclerosis, and Parkinson's istered in the absence of permeability enhancers, Some of disease. which have neurostimulatory effects themselves. In one embodiment of the methods described above, the In one aspect, the disclosure provides a method for CNS disorder is Alzheimer's disease. treating a central nervous system (CNS) disorder in a subject 30 in need thereof, the method including delivering a therapeu In one embodiment of the methods described above, tically effective amount of a composition comprising pooled intranasal administration of the composition includes the use human immunoglobulin G (IgG) to the brain of the subject, of a non-invasive intranasal delivery device. where delivering the composition to the brain includes In one embodiment of the methods described above, intranasally administering the composition directly to the 35 intranasal administration of the composition includes olfactory epithelium of the nasal cavity of the subject. administration of a liquid drop of the composition directly In another aspect, the disclosure provides a method for onto the nasal epithelium, the nasal epithelium of the subject treating a central nervous system (CNS) disorder in a subject associated with trigeminal nerve endings, or the upper third in need thereof, the method including delivering a therapeu of the nasal cavity of the subject. tically effective amount of a composition comprising pooled 40 In one embodiment of the methods described above, human immunoglobulin G (IgG) to the brain of the subject, intranasal administration of the composition includes where delivering the composition to the brain includes directed administration of an aerosol of the composition to intranasally administering the composition to a nasal epi the nasal epithelium, the nasal epithelium of the subject thelium of the subject associated with trigeminal nerve associated with trigeminal nerve endings, or the upper third endings. 45 of the nasal cavity of the subject. In another aspect, the disclosure provides a method for In one embodiment of the methods described above, the treating a central nervous system (CNS) disorder in a subject aerosol of the composition is a liquid aerosol. in need thereof, the method including delivering a therapeu In one embodiment of the methods described above, the tically effective amount of a composition comprising pooled aerosol of the composition is a powder aerosol. human immunoglobulin G (IgG) to the brain of the subject, 50 In one embodiment of the methods described above, at where delivering the composition to the brain includes least 40% of the pooled human IgG administered to the intranasally administering the composition to the upper third Subject contacts the nasal epithelium of the Subject, the of the nasal cavity of the subject. olfactory epithelium of the nasal cavity of the subject, a In another aspect, the disclosure provides a method for nasal epithelium of the Subject associated with trigeminal treating a central nervous system (CNS) disorder in a subject 55 nerve endings, the upper third of the nasal cavity of the in need thereof, the method including delivering a therapeu subject, or one or both maxillary sinus of the subject. tically effective amount of a composition comprising pooled In one embodiment of the methods described above, at human immunoglobulin G (IgG) to the brain of the subject, least 50% of the pooled human IgG administered to the where delivering the composition to the brain includes Subject contacts the olfactory epithelium of the nasal cavity intranasally administering the composition to one or both 60 of the subject, the nasal epithelium of the subject associated maxillary sinus of the Subject. with trigeminal nerve endings, the upper third of the nasal In one embodiment of the methods described above, the cavity of the subject, or one or both maxillary sinus of the CNS disorder is selected from the group consisting of a Subject. neurodegenerative disorder of the central nervous system, a In one embodiment of the methods described above, at systemic atrophy primarily affecting the central nervous 65 least 60% of the pooled human IgG administered to the system, an extrapyramidal and , a demy Subject contacts the olfactory epithelium of the nasal cavity elinating disorder of the central nervous system, an episodic of the subject, the nasal epithelium of the subject associated US 9,556.260 B2 10 with trigeminal nerve endings, the upper third of the nasal In one embodiment of the methods described above, the cavity of the subject, or one or both maxillary sinus of the method includes intranasally administering to the Subject a Subject. dose of pooled human IgG at least twice monthly. In a In one embodiment of the methods described above, the specific embodiment of the methods described above, the pooled human IgG composition does not contain a perme method includes intranasally administering to the Subject a ability enhancer. dose of pooled human IgG at least once weekly. In a specific In one embodiment of the methods described above, the embodiment of the methods described above, the method pooled human IgG composition consists essentially of includes intranasally administering to the Subject a dose of pooled human IgG and an amino acid. pooled human IgG at least twice weekly. In a specific In one embodiment of the methods described above, the 10 embodiment of the methods described above, the method amino acid is selected from the group consisting of glycine, includes intranasally administering to the Subject a dose of histidine, and proline. In a specific embodiment of the pooled human IgG at least once daily. In a specific embodi methods provided above, the amino acid is glycine. ment of the methods described above, the method includes In one embodiment of the methods described above, the 15 intranasally administering to the Subject a dose of pooled pooled human IgG composition is an aqueous composition. human IgG at least twice daily. In one embodiment of the methods described above, the In one embodiment of the methods described above, the pooled human IgG composition includes from 10 mg/mL to pooled human IgG composition includes at least 0.1% 250 mg/mL pooled human IgG and from 50 mM to 500 mM anti-amyloid B IgG. glycine. In one embodiment of the methods described above, the In one embodiment of the methods described above, the method further includes administering a second therapy for pH of the composition is from 4.0 to 6.0. In another the CNS disorder to the subject in need thereof. embodiment of the methods provided above, the pH of the In one embodiment of the methods described above, the composition is from 4.0 to 7.5. In another embodiment of the second therapy for the CNS disorder is a cholinesterase methods provided above, the pH of the composition is from 25 inhibitor. In a specific embodiment of the methods described 6.0 to 7.5. above, the cholinesterase inhibitor is selected from the group In one embodiment of the methods described above, the consisting of donepezil (e.g., ARICEPTR), rivastigmine pooled human IgG composition is a dry powder composi (e.g., EXELONR), galantamine (e.g., RAZADYNER), and tion. tacrine (e.g., COGNEX(R). In one embodiment of the methods described above, the 30 In one embodiment of the methods described above, the dry powder composition is prepared from an aqueous solu second therapy for the CNS disorder is an inhibitor of tion including from 10 mg/mL to 250 mg/mL pooled human NMDA-type glutamate receptor. In a specific embodiment IgG and from 50 mM to 500 mM glycine. of the methods described above, the inhibitor of NMDA In one embodiment of the methods described above, the type glutamate receptor is memantine. dry powder composition is prepared from an aqueous solu 35 In another aspect, the disclosure provides the use of a tion having a pH of from 4.0 to 6.0. In another embodiment composition comprising pooled human immunoglobulin G of the methods provided above, the pH of the composition (IgG) for the treatment of a central nervous system (CNS) is from 4.0 to 7.5 In another embodiment of the methods disorder in a subject in need thereof by intranasal adminis provided above, the pH of the composition is from 6.0 to 7.5 tration. In one embodiment of the methods described above, the 40 In some embodiments of the uses described above, intra method includes intranasally administering to the Subject a nasal administration includes administration to the nasal dose of from 0.08 mg to 100 mg pooled human IgG per kg epithelium of the subject. In other embodiments of the uses body weight of the Subject (mg IgG/kg). In a specific described above, intranasal administration comprises embodiment of the methods provided above, the method administration to the olfactory epithelium of the nasal cavity includes intranasally administering to the Subject a dose of 45 of the subject. In other embodiments of the uses described from 0.2 mg to 40 mg pooled human IgG per kg body weight above, intranasal administration includes administration to a of the Subject (mg IgG/kg). In a specific embodiment of the nasal epithelium of the Subject associated with trigeminal methods provided above, the method includes intranasally nerve endings. In other embodiments of the uses described administering to the Subject a dose of from 5 mg to 20 mg above, intranasal administration includes administration to pooled human IgG per kg body weight of the Subject (mg 50 the upper third of the nasal epithelium of the nasal cavity of IgG/kg). In a specific embodiment of the methods provided the subject. In yet other embodiments, of the uses described above, the method includes intranasally administering to the above, intranasal administration includes administration to Subject a dose of from 5 mg to 10 mg pooled human IgG per one or both maxillary sinus of the subject. kg body weight of the Subject (mg IgG/kg). In a specific In one embodiment of the uses described above, the CNS embodiment of the methods provided above, the method 55 disorder is selected from the group consisting of a neuro includes intranasally administering to the Subject a dose of degenerative disorder of the central nervous system, a sys from 1 mg to 5 mg pooled human IgG per kg body weight temic atrophy primarily affecting the central nervous sys of the Subject (mg IgG/kg). tem, an extrapyramidal and movement disorder, a In one embodiment of the methods described above, the demyelinating disorder of the central nervous system, an method includes intranasally administering to the Subject a 60 episodic or paroxysmal disorder of the central nervous fixed dose of from 50 mg to 10 g pooled human IgG. In a system, a paralytic syndrome of the central nervous system, specific embodiment of the methods provided above, the a nerve, nerve root, or plexus disorder of the central nervous method includes intranasally administering to the Subject a system, an organic mental disorder, a mental or behavioral fixed dose of from 100 mg to 5.0 g pooled human IgG. In a disorder caused by psychoactive Substance use, a schizo specific embodiment of the methods provided above, the 65 phrenia, Schizotypal, or delusional disorder, a mood (affec method includes intranasally administering to the Subject a tive) disorder, neurotic, stress-related, or Somatoform disor fixed dose of from 500 mg to 2.5g pooled human IgG. der, a behavioral syndrome, an adult personality or behavior US 9,556.260 B2 11 12 disorder, a psychological development disorder, and a child histidine, and proline. In a specific embodiment of the onset behavioral or emotional disorder. methods provided above, the amino acid is glycine. In one embodiment of the uses described above, the CNS In one embodiment of the uses described above, the disorder is selected from the group consisting of Alzheim pooled human IgG composition is an aqueous composition. er's disease, Parkinson's disease, multiple Sclerosis, amyo In one embodiment of the uses described above, the trophic lateral Sclerosis (ALS), Huntington's disease, cere pooled human IgG composition includes from 10 mg/mL to bral palsy, bipolar disorder, schizophrenia, and Pediatric 250 mg/mL pooled human IgG and from 50 mM to 500 mM acute-onset neuropyschiatric syndrome (PANS). glycine. In one embodiment of the uses described above, the CNS In one embodiment of the uses described above, the pH of disorder is selected from the group consisting of Alzheim 10 er's disease, Parkinson's disease, multiple sclerosis, Pediat the composition is from 4.0 to 6.0. In another embodiment ric Autoimmune Neuropsychiatric Disorders Associated of the uses described above, the pH of the composition is with Streptococcal infections (PANDAS), and Pediatric from 4.0 to 7.5. In another embodiment of the methods acute-onset neuropyschiatric syndrome (PANS). provided above, the pH of the composition is from 6.0 to 7.5. In one embodiment of the uses described above, the CNS 15 In one embodiment of the uses described above, the disorder is selected from the group consisting of Alzheim pooled human IgG composition is a dry powder composi er's disease, multiple Sclerosis, and Parkinson's disease. tion. In one embodiment of the uses described above, the CNS In one embodiment of the uses described above, the dry disorder is Alzheimer's disease. powder composition is prepared from an aqueous Solution In one embodiment of the uses described above, intrana including from 10 mg/mL to 250 mg/mL pooled human IgG sal administration of the composition includes the use of a and from 50 mM to 500 mM glycine. non-invasive intranasal delivery device. In one embodiment of the uses described above, the dry In one embodiment of the uses described above, intrana powder composition is prepared from an aqueous Solution sal administration of the composition includes administra having a pH of from 4.0 to 6.0. In another embodiment of tion of a liquid drop of the composition directly onto the 25 the uses described above, the pH of the composition is from nasal epithelium, the nasal epithelium of the Subject asso 4.0 to 7.5 In another embodiment of the uses described ciated with trigeminal nerve endings, or the upper third of above, the pH of the composition is from 6.0 to 7.5 the nasal cavity of the subject. In one embodiment of the uses described above, the use In one embodiment of the uses described above, intrana includes intranasally administering to the Subject a dose of sal administration of the composition includes directed 30 from 0.08 mg to 100 mg pooled human IgG per kg body administration of an aerosol of the composition to the nasal weight of the Subject (mg IgG/kg). In a specific embodiment epithelium, the nasal epithelium of the subject associated of the uses described above, the use includes intranasally with trigeminal nerve endings, or the upper third of the nasal administering to the Subject a dose of from 0.2 mg to 40 mg cavity of the subject. pooled human IgG per kg body weight of the Subject (mg In one embodiment of the uses described above, the 35 IgG/kg). In a specific embodiment of the uses described aerosol of the composition is a liquid aerosol. above, the use includes intranasally administering to the In one embodiment of the uses described above, the Subject a dose of from 5 mg to 20 mg pooled human IgG per aerosol of the composition is a powder aerosol. kg body weight of the Subject (mg IgG/kg). In a specific In one embodiment of the uses described above, at least embodiment of the uses described above, the use includes 40% of the pooled human IgG administered to the subject 40 intranasally administering to the Subject a dose of from 5 mg contacts the nasal epithelium of the subject, the olfactory to 10 mg pooled human IgG per kg body weight of the epithelium of the nasal cavity of the Subject, a nasal epithe Subject (mg IgG/kg). In a specific embodiment of the uses lium of the Subject associated with trigeminal nerve endings, described above, the use includes intranasally administering the upper third of the nasal cavity of the subject, or one or to the Subject a dose of from 1 mg to 5 mg pooled human both maxillary sinus of the subject. 45 IgG per kg body weight of the Subject (mg IgG/kg). In one embodiment of the uses described above, at least In one embodiment of the uses described above, the use 50% of the pooled human IgG administered to the subject includes intranasally administering to the Subject a fixed contacts the nasal epithelium of the subject, the olfactory dose of from 50 mg to 10g pooled human IgG. In a specific epithelium of the nasal cavity of the Subject, a nasal epithe embodiment of the uses provided above, the use includes lium of the Subject associated with trigeminal nerve endings, 50 intranasally administering to the Subject a fixed dose of from the upper third of the nasal cavity of the subject, or one or 100 mg to 5.0 g pooled human IgG. In a specific embodi both maxillary sinus of the subject. ment of the uses provided above, the use includes intrana In one embodiment of the uses described above, at least sally administering to the subject a fixed dose of from 500 60% of the pooled human IgG administered to the subject mg to 2.5g pooled human IgG. contacts the nasal epithelium of the subject, the olfactory 55 In one embodiment of the uses described above, the epithelium of the nasal cavity of the Subject, a nasal epithe method includes intranasally administering to the Subject a lium of the Subject associated with trigeminal nerve endings, dose of pooled human IgG at least twice monthly. In a the upper third of the nasal cavity of the subject, or one or specific embodiment of the uses described above, the both maxillary sinus of the subject. method includes intranasally administering to the Subject a In one embodiment of the uses described above, the 60 dose of pooled human IgG at least once weekly. In a specific pooled human IgG composition does not contain a perme embodiment of the uses described above, the method ability enhancer. includes intranasally administering to the Subject a dose of In one embodiment of the uses described above, the pooled human IgG at least twice weekly. In a specific pooled human IgG composition consists essentially of embodiment of the uses described above, the method pooled human IgG and an amino acid. 65 includes intranasally administering to the Subject a dose of In one embodiment of the uses described above, the pooled human IgG at least once daily. In a specific embodi amino acid is selected from the group consisting of glycine, ment of the uses described above, the method includes US 9,556.260 B2 13 14 intranasally administering to the Subject a dose of pooled rescent staining of amyloid plaques in the hippocampus and human IgG at least twice daily. cortex of aged TG2576 transgenic mice (field of view=5.3 In one embodiment of the uses described above, the mm). For the immunofluorescent staining, mice were intra pooled human IgG composition includes at least 0.1% nasally administered saline, low dose IgG, or high dose IgG anti-amyloid B IgG. three times weekly over a period of 8 months. Each value is In one embodiment of the uses described above, the reported as the mean value for the cohorts-tstandard error. method further includes administering a second therapy for FIG. 4 illustrates a Kaplan-Meier curve for survival rates the CNS disorder to the subject in need thereof. of transgenic and wild-type mice administered IgG intrana In one embodiment of the uses described above, the sally. These mice belong to a different cohort than the mice second therapy for the CNS disorder is a cholinesterase 10 used for plaque analysis in FIG. 3. inhibitor. In a specific embodiment of the uses described FIG. 5 Illustrates the seven coronal brain slices which above, the cholinesterase inhibitor is selected from the group were hemisected from intranasal 'I IgG treated rats used to consisting of donepezil (e.g., ARICEPTR), rivastigmine assess CNS delivery in Example 8. (e.g., EXELONR), galantamine (e.g., RAZADYNE(R), or FIGS. 6A-6B show comparative results of the intactness tacrine (e.g., COGNEX(R). 15 of IgG sprayed through a device designed for intranasal In one embodiment of the uses described above, the delivery with that of non-sprayed control. FIG. 6A shows a second therapy for the CNS disorder is an inhibitor of Coomassie stained, non-reducing gel of sprayed and non NMDA-type glutamate receptor. In a specific embodiment sprayed (control). IgG. FIG. 6B shows a Western blot of a of the uses described above, the inhibitor of NMDA-type reducing gel probed with an anti-IgG antibody. glutamate receptor is memantine. FIG. 7 shows results demonstrating the highly efficient olfactory epithelium targeting of IN device administration in BRIEF DESCRIPTION OF DRAWINGS rats. The upper panel shows the deposition of IN IgG after device administration of 15 uL of 25% IVIG solution spiked The patent or application file contains at least one drawing with 0.01% fluorescein tracer in a rat. The lower panel executed in color. Copies of this patent or patent application 25 shows the deposition pattern after deposition of 15 uL of publication with color drawing(s) will be provided by the 25% IVIG solution spiked with 0.01% fluorescein tracer Office upon request and payment of the necessary fee. administered via nose drops. OB-olfactory bulb, FIGS. 1A-1F show brain slices from rats used to assess OE-olfactory epithelium, RE-respiratory epithelium, the biodistribution of intranasally administered IgG in NS=naris. Example 2. Six 2 mm slices (3 rostral to the optic chiasm and 30 FIGS. 8A-8C illustrate data showing a decrease of amy 3 caudal) were acquired. loid load in the low IgG and high IgG intranasal treatment FIG.1G illustrates a brain bisected along the midline. The groups. FIG. 8A shows the total amyloid area (plaque and bisected brain is further dissected in to midbrain, pons, vasculature). FIG. 8B shows the number (ii) of amyloid medulla, and cerebellum for biodistribution analysis. deposits (plaque and vasculature). FIG. 8C shows the total FIGS. 2A-2B illustrate results of average brain tissue 125 35 intensity of all amyloid deposits (i.e., the Sum Intensity). IgG concentrations (nM) 30 and 90 minutes after intranasal FIGS. 9A-9C illustrate data showing a decrease in amy administration of IgG. FIG. 2A illustrates results of brain loid is a result of a decrease in plaque load. FIG. 9A shows tissue 'I IgG concentrations (nM) 30 (n=8) and 90 (n-6) the total amyloid area (plaque and vasculature). FIG. 9B minutes after administration of a liquid protein IgG prepa shows the number (ii) of amyloid deposits (plaque and ration, normalized to a 6.0 mg dose. FIG. 2B illustrates 40 vasculature). FIG. 9C shows the total intensity of all amy results of brain tissue 'I IgG concentrations (nM) 30 loid deposits (i.e., the Sum Intensity). (n=12) and 90 (n-6) minutes after administration of a solid FIGS. 10A-10C illustrate data showing that the vascular microsphere IgG preparation, normalized to a 6.0 mg dose. component of the amyloid was found to increase slightly FIGS. 3A-3E illustrate IHC data on cortical and hip when a decrease in amyloid as a result of a decrease in pocampal brain slices. Plaque content was determined for 12 45 plaque load was observed (FIG. 9). FIG. 10A shows the mice from each cohort (WT-Saline, WT-High, TG-Saline, vascular amyloid area. FIG. 10B shows the number (ii) of TG-Low, and TG-High; shown left to right in the charts, vascular deposits. FIG. 10C shows the total intensity of respectively). FIG. 3A shows the percent area covered by vascular deposits (i.e., the Sum Intensity). beta-amyloid plaques. Four slides from the cortex of each FIGS. 11A-11B illustrate data showing the relative pro mouse were analyzed using Image.J Software. The data is 50 portions of vascular and plaque amyloid as it contributes to distributed in the given range by plaque radius size (in total amyloid. FIG. 11A shows the relative plaque contri micrometers). Significant differences between transgenic bution to total amyloid. FIG. 11B shows the relative vascular treatment groups are marked in the graph with the p-value. contribution to total amyloid. FIG. 3B shows the average number of beta-amyloid plaques. FIGS. 12A-12F show Congo Red stained sagittal sections Four slides from the cortex of each mouse were analyzed 55 captured with confocal fluorescent microscopy. FIG. 12A using Image.J Software. The data is distributed in the given shows a Z-Stack max intensity projection image created from range by plaque radius size (in micrometers). Significant five individual images at 10x with a 512x512 resolution. differences between transgenic treatment groups are marked FIGS. 12B-12F show single images created from thirty of in the graph with the p-value. FIG. 3C shows the average Z-Stacks projections as shown in FIG. 12A, encompassing number of beta-amyloid plaques. Four slides from the hip 60 the whole tissue section that were tiled (6x5, 5% overlap). pocampus of each mouse were analyzed using Image.J Representative images from the groups: Tg-Saline with Software. The data is distributed in the given range by plaque Thresholding, Full-Resolution, Portion of the cortex and radius size (in micrometers). FIG. 3D shows the percent area hippocampus (FIG. 12A): Tg-Low without Thresholding covered by beta-amyloid plaques. Four slides from the (FIG. 12B); WT-Saline with thresholding (FIG. 12C): Tg hippocampus of each mouse were analyzed using Image.J 65 Saline with thresholding (FIG. 12D); Tg-Low with thresh Software. The data is distributed in the given range by plaque olding (FIG. 12E); and Tg-High with thresholding (FIG. radius size (in micrometers). FIG. 3E shows immunofluo 12F). US 9,556.260 B2 15 16 FIGS. 13 A-13B illustrate data for the average staining is capable of increasing lifespan in the Alzheimer's mouse intensity for the Astrocyte marker GFAP (FIG. 13A) and the model, indicating efficacy in Alzheimer's treatment. microglial marker CD11b (FIG. 13B). Moreover, intranasal administration of IgG significantly FIG. 14 is an example image of amyloid (blue), GFAP reduced plaques in the cerebral cortex of in the Alzheimer's (green) and CD11b (red) staining from a Tg2576 mouse mouse model. It is shown in Example 4 that treatment with brain that had been treated with a high dose of IN IgG. pooled human IgG reduced the percent area covered by CD11b staining was often observed surrounding the amyloid plaques in the Alzheimer's mouse model by about 25%, plaques. when administered intranasally at either low (0.4 mg/kg/2 wk; p=0.014) or high (0.8 mg/kg/2 wk; p=0.037) dosage. DETAILED DESCRIPTION OF INVENTION 10 This is further indication of the efficacy of intranasal admin istration of IgG in the treatment of Alzheimer's disease. Introduction As further demonstrated herein, intranasal administration The present disclosure provides methods and composi results in a much more discriminate delivery of pooled tions for treating a central nervous system (CNS) disorder in human IgG to the brain, as compared to intravenous admin 15 istration. For example, it is shown in Example 9 that a subject by intranasal delivery of a therapeutically effective intranasal administration of pooled human immunoglobulin amount of pooled human immunoglobulin G (IgG) directly G resulted in a 6-fold lower blood exposure as compared to to the epithelium of the nasal cavity of the subject. In a intravenous administration. The lower system exposure of specific embodiment, pooled human IgG is administered IgG provided by intranasal administration advantageously directly to the olfactory epithelium of the nasal cavity. In reduces the risk of side effects associated with the systemic Some embodiments, pooled IgG is delivered to the upper exposure of IgG. third of the nasal cavity, e.g., above the lower turbinates. In Advantageously, it was also found that pooled human some embodiments, pooled IgG is delivered to the brain via immunoglobulin G was efficiently delivered to the brain the trigeminal nerve after intranasal administration to the following intranasal administration in the absence of a nasal respiratory epithelium. In a specific embodiment, 25 permeability enhancer (e.g., membrane fluidizers, tight junc pooled IgG is delivered to the brain via the maxillary nerve tion modulators, and medium chain length fatty acids and after intranasal administration to the nasal respiratory epi salts and esters thereof, as described below). Previous thelium. In other embodiments, pooled IgG is delivered to reports have Suggested that in order to achieve efficient the brain after administration to the maxillary sinus. transport of biotherapeutics (e.g., mimetibodies and Fc In some embodiments, methods and compositions for the 30 fusions) through the olfactory epithelium, a permeability treatment of Alzheimer's disease, multiple Sclerosis, and enhancer is required (WO 2009/058957). However, as Parkinson's disease via intranasal administration of pooled shown in the examples provided herein, pooled human IgG human IgG are provided herein. In other embodiments, the is efficiently delivered to the brain when intranasally admin methods and compositions provided herein are useful for the istered as a liquid or dry powder formulated with only an treatment of CNS disorder known to one of skill in the art 35 amino acid (e.g., glycine). including, without limitation, a neurodegenerative disorder Advantageously, it is also shown herein that the dose of of the central nervous system, a systemic atrophy primarily pooled human IgG can be significantly reduced when affecting the central nervous system, an extrapyramidal and administered intranasally, as compared to intravenous movement disorder, a demyelinating disorder of the central administration. For example, it is shown in Example 9 that nervous system, an episodic or paroxysmal disorder of the 40 administration of a low dose of pooled human IgG (0.002 central nervous system, a paralytic syndrome of the central g/kg IgG) intranasally delivered directly to the olfactory nervous system, a nerve, nerve root, or plexus disorder of the epithelium results in Substantially the same amount of IgG central nervous system, an organic mental disorder, a mental being delivered to the right and left hemispheres of the brain or behavioral disorder caused by psychoactive substance as for intravenous administration of a ten-fold higher dose of use, a Schizophrenia, Schizotypal, or delusional disorder, a 45 pooled human IgG (0.02 g/kg IgG, compare corrected AUC mood (affective) disorder, neurotic, stress-related, or Soma values for right and left hemisphere IgG delivery in Table 71 toform disorder, a behavioral syndrome, an adult personality and Table 72). A ten-fold reduction in the amount of pooled or behavior disorder, a psychological development disorder, human IgG required for administration is significant because or a child onset behavioral or emotional disorder. In some of the limited Supply of pooled human IgG and the high cost embodiments, the CNS disorder is selected from the group 50 associated therewith. consisting of Alzheimer's disease, Parkinson's disease, mul The results described above, which taken together Suggest tiple Sclerosis, amyotrophic lateral Sclerosis (ALS), Hun that low doses of intranasally administered pooled human tington's disease, cerebral palsy, bipolar disorder, Schizo IgG is effective for the treatment of Alzheimer's disease, are phrenia, or Pediatric acute-onset neuropySchiatric syndrome Surprising given the difficulty of delivering full-length (PANS). In some embodiments, the CNS disorder is selected 55 immunoglobulins to the brain via intranasal administration. from the group consisting of Alzheimer's disease, Parkin First, although antibody fragments (e.g., Fabs) have previ son's disease, multiple Sclerosis, Pediatric Autoimmune ously been administered intranasally, the inventors are Neuropsychiatric Disorders Associated with Streptococcal unaware of any reports demonstrating delivery of full-length infections (PANDAS), or Pediatric acute-onset neuropyschi antibodies to the brain via intranasal administration. In fact, atric syndrome (PANS). 60 it has been reported that the delivery of full-length antibod Advantageously, it is shown herein that intranasal admin ies poses a great difficulty in the field of medicine (Harmsen istration of IgG increased weight and Survival time of M Met al., Appl Microbiol Biotechnol., 2007, 77(1): 13-22: Alzheimer's disease mice models. For example, it is shown Athwal G S, Innovations in Pharmaceutical Technology, in Example 6 that intranasal administration of IgG, at either July 2009; WO 2006/091332; and WO 2009/058957). Con high (0.8 g/kg once every two weeks) or low (0.4 g/k once 65 sistent with these reports, Applicants found that antibody every two weeks) doses, prolonged the lifespan of TG2576 fragments are delivered much more readily to the brain, as mice. This result shows that intranasal administration of IgG compared to full-length immunoglobulins, after intranasal US 9,556.260 B2 17 18 administration. For example, it is shown in Example 2 that, include many neurodegenerative diseases (e.g., Hunting on average, the concentration of Fabs in brain tissue post ton's disease, Amyotrophic lateral Sclerosis (ALS), heredi intranasal administration is 19-times higher than the con tary spastic hemiplegia, primary lateral Sclerosis, spinal centration of full-length immunoglobulins post-intranasal muscular atrophy, Kennedy's disease, Alzheimer's disease, administration. Given the significantly lower delivery of 5 ataxias, Huntington's disease, Lewy body disease, a poly full-length immunoglobulins to the brain, it is Surprising that glutamine repeat disease, and Parkinson's disease) and psy intranasal administration of pooled immunoglobulins pro chiatric disorders (e.g., mood disorders, Schizophrenias, and vides the effective results shown herein. autism). Non-limiting examples of ataxia include Friedre Advantageously, intranasal delivery of the pooled human ich's ataxia and the spinocerebellar ataxias. Specifically for IgG composition disclosed herein can be accomplished by a 10 this application, CNS disorders do not include disorders non-invasive means, as compared to intravenous, Subcuta resulting from acute viral and bacterial infections. neous, and intramuscular administration, all of which Non-limiting examples of CNS disorders include neuro require puncture of the skin of the subject. For example, it degenerative disorders of the central nervous system, sys is shown in Example 3 that pooled human IgG can be temic atrophies primarily affecting the central nervous sys efficiently delivered to the brain using nasal drops or a nasal 15 tem, extrapyramidal and movement disorders, Spray. demyelinating disorders of the central nervous system, epi Another benefit provided by the methods and composi sodic or paroxysmal disorders of the central nervous system, tions provided herein for intranasal administration of pooled paralytic syndromes of the central nervous system, nerve, human IgG is improved patient compliance. Treatment with nerve root, or plexus disorders of the central nervous system, intravenous IgG (IVIG) requires a lengthy administration organic mental disorders, mental or behavioral disorders period under medical Supervision, generally taking place at caused by psychoactive Substance use, Schizophrenic, hospitals and medical facilities. For example, initial admin Schizotypal, or delusional disorders, mood (affective) disor istration of IVIG occurs over a 2 to 5 hour period, once a day ders, neurotic, stress-related, or somatoform disorders, for 2 to 7 consecutive days. Follow-up doses, also typically behavioral syndromes, adult personality or behavior disor administered at a hospital over a period of 2 to 5 hours, are 25 ders, psychological development disorders, and child onset required every 1 to 4 weeks depending on the indication behavioral or emotional disorders. (Diagnostic and Statisti being treated and dosing regimen. Such an administration cal Manual of Mental Disorders, 4th Edition (DSM-IV): The regime is time consuming and inconvenient for many World Health Organization, The International Classification patients. In comparison, intranasal administration can be of Diseases, 10th revision (ICD-10), Chapter V. Further administered at home without medical Supervision. Also, 30 exemplary CNS disorders are provided herein below. intranasal administration can be performed quickly, over Neurodegenerative CNS disorders are typically charac several minutes depending on the number of drops/sprays terized by progressive dysfunction and/or cell death in the required, rather than several hours as required for intrave central nervous system. The hallmark of many neurodegen nous administration. Thus, treatment can be prescribed more erative CNS disorders is the accumulation of misfolded frequently at lower doses to maintain an effective level of 35 proteins, such as beta-amyloid, tau, alpha-synuclein, and IgG in the CNS with minimal inconvenience because admin TDP-43, both intracellularly and extracellularly. Many neu istration occurs at home in a shorter period of time. rodegenerative diseases are also associated with gross mito Furthermore, IVIG therapy requires catheterization which chondrial dysfunction. Common examples of neurodegen can cause discomfort and infection at the site of the catheter. erative CNS disorders include Alzheimer's disease (AD), IVIG Solutions are often high in Sodium and glucose to 40 Parkinson's disease (PD), Huntington's disease, and Amyo create isotonicity, causing increased risk to the elderly trophic lateral sclerosis (ALS). population, which already have increased rates of diabetes Psychiatric disorders (also referred to as mental illnesses) and high blood pressure. On the other hand, intranasal commonly present with cognitive deficits and mood dys administration is non-invasive, i.e. there is no catheteriza regulation. Psychiatric disorders are generally defined by a tion and does not carry invasive-procedure related risks Such 45 combination of how a person feels, acts, thinks or perceives. as infection and discomfort at the site of the catheter. Well established systems for the classification of psychiatric Intranasal administration of pooled human IgG composi disorders include the International Statistical Classification tions is an improved procedure for elderly persons because of Diseases and Related Health Problems, 10th Revision it does not require IV perfusion and thus does not create a (World Health Organization, tenth revision (2010), the con systemic increase in concentrations of salt or glucose in the 50 tent of which is hereby expressly incorporated by reference blood. in its entirety for all purposes) and the Diagnostic and Thus, as compared to currently utilized modes of admin Statistical Manual of Mental Disorders (DSM-IV: American istering pooled human IgG (e.g., intravenous, Subcutaneous, Psychiatric Association, DS-IV-TR (2000), the content of and intramuscular) intranasal administration increases the which is hereby expressly incorporated by reference in its ease of administration, decreases overall administration 55 entirety for all purposes). Common examples of psychiatric time, decreases the number of hospital visits required, and disorders include mood disorders, Schizophrenia, and eliminates the risks associated with catheter-based admin autism. istration (e.g., IV administration). Thus, implementation of As used herein, the terms “pooled human immunoglobu intranasal administration of pooled human IgG will result in lin G' and “pooled human IgG” refer to a composition improved patient compliance. 60 containing polyvalent immunoglobulin G (IgG) purified Definitions from the blood/plasma of multiple donors, e.g., more than a As used herein, the terms “disorder of the central nervous hundred or more than a thousand blood donors. Typically, system.” “central nervous system disorder,” “CNS disor the composition will be at least 80% IgG (w/w, e.g., weight der,’ and the like refer to a disorder affecting either the IgG per weight total protein), preferably at least 85%. 86%, spinal cord (e.g., a ) or brain (e.g., an encepha 65 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, lopathy) of a subject, which commonly presents with neu 97%, 98%, or 99% IgG (w/w). In certain embodiments, the rological and/or psychiatric symptoms. CNS disorders pooled human IgG composition contains intact IgG immu US 9,556.260 B2 19 20 noglobulins. In other embodiments, the pooled human IgG 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, composition contains IgG fragments, for example those 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, prepared by treatment of intact antibodies with trypsin. In 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, certain embodiments, the pooled human IgG compositions 3.5%, 4.0%, 4.5%, 5.0% or more anti-CAPS IgG. used in the treatments disclosed herein contain natural or In one embodiment, a high titer anti-amyloid B pooled synthetic modifications, e.g., post-translational modifica immunoglobulin G composition contains at least 0.02% tions and/or chemical modifications. anti-amyloid B monomer IgG. In another embodiment, a As used herein, the terms “high titer anti-amyloid f high titer anti-amyloid 3 pooled immunoglobulin G com pooled immunoglobulin G' and “high titer anti-amyloid B position contains at least 0.04% anti-amyloid B monomer pooled IgG refer to a composition containing polyvalent 10 IgG. In yet other embodiments, a high titer anti-amyloid B immunoglobulin G (IgG) purified from the blood/plasma of pooled immunoglobulin G composition contains at least multiple donors, e.g., more than a hundred or more than a 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, thousand blood donors, having a relative titer of anti 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, amyloid B immunoglobulin G that is greater than the 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, expected titer of anti-amyloid B immunoglobulins in a 15 0.85%, 0.9%, 0.95%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, pooled IgG composition prepared from the blood/plasma of 4.0%, 4.5%, 5.0% or more anti-amyloid B monomer IgG. more than a thousand random individuals. Commercially High titer anti-amyloid 3 pooled IgG can be prepared available intravenous immunoglobulin G (IVIG) prepara according to standard methods for the manufacture of tions contain IgGs that specifically recognize epitopes of pooled IgG starting with a standard pool of blood/plasma of various conformers of amyloid B, e.g., amyloid B monomers, multiple donors, e.g., more than a hundred or more than a amyloid B fibrils, and cross-linked amyloid 3 protein species thousand blood donors, and Subsequently enriched for anti (CAPS). It has been reported that a commercial preparation amyloid 3 immunoglobulin G. Methods for the enrichment of GAMMAGARD LIQUIDR) (10% Immune Globulin of target-specific immunoglobulin G molecules are well Infusion (Human); Baxter International Inc., Deerfield, Ill.) known in the art (for example, see U.S. Patent Application contains 0.1% anti-amyloid B fibril IgG, 0.04% anti-CAPS 25 Publication No. 2004/0101909, the content of which is IgG, and 0.02% anti-amyloid B monomer IgG, having ECso hereby expressly incorporated by reference herein in its affinities of 40 nM, 40 nM, and 350 nM for their target entirety for all purposes). Alternatively, high titer anti amyloid f conformer, respectively (O'Nuallain B. et al., amyloid B pooled IgG can be prepared according to standard Biochemistry, 2008 Nov. 25; 47(47): 12254-6, the content of methods for the manufacture of pooled IgG starting with an which is hereby incorporated by reference in its entirety for 30 enriched pool of blood/plasma from at least fifty, one all purposes). In some embodiments, a high titer anti hundred, two hundred, five hundred, or one thousand donors amyloid B pooled immunoglobulin G composition contains having a high relative titer of anti-amyloid B immunoglobu a high titer of IgG specific for one or more conformer of lin G. As compared to the manufacture of standard IgG for amyloid 3. In other embodiments, a high titer anti-amyloid intravenous administration, hyperimmune IgG preparations B pooled immunoglobulin G composition contains a high 35 are commonly prepared from Smaller donor pools. These titer of IgG specific for amyloid B monomers, amyloid B enriched pools of blood/plasma can be formed, for example, fibrils, and cross-linked amyloid f protein species (CAPS). by selectively pooling blood/plasma donations or donors Accordingly, in one embodiment, a high titeranti-amyloid with a high relative titer of anti-amyloid B immunoglobulin B pooled immunoglobulin G composition contains at least G, e.g., by selection of high titer blood/plasma donations or 0.1% anti-amyloid B IgG (e.g., 0.1% IgG with specific 40 donors. Alternatively, an enriched pool of blood/plasma can affinity for any amyloid 3 conformer). In another embodi be formed by Screening for blood/plasma donations or ment, a high titer anti-amyloid B pooled immunoglobulin G donors with a low relative titer of anti-amyloid 3 immuno composition contains at least 0.2% anti-amyloid B IgG. In globulin G and excluding these donations or donors from the yet other embodiments, a high titer anti-amyloid B pooled starting blood/plasma pool, e.g., Screening for low titer immunoglobulin G composition contains at least 0.15%, 45 blood/plasma donations or donors. 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, As used herein, the term “intactness” refers to a percent 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, age of therapeutic agent that has not been at least partially 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or degraded at a particular point in time following administra more anti-amyloid B IgG. tion. In one embodiment, intactness is a measure of the total In one embodiment, a high titer anti-amyloid B pooled 50 administered dose of the therapeutic agent that has not been immunoglobulin G composition contains at least 0.1% anti at least partially degraded at the particular point in time (i.e., amyloid B fibril IgG. In another embodiment, a high titer systemic intactness). In another embodiment, intactness is a anti-amyloid 3 pooled immunoglobulin G composition con measure of the therapeutic agent present at a particular site tains at least 0.2% anti-amyloid B fibril IgG. In yet other of the Subject, e.g., brain or bloodstream, which has not been embodiments, a high titer anti-amyloid B pooled immuno 55 at least partially degraded (i.e., local intactness). In one globulin G composition contains at least 0.15%, 0.2%, embodiment, the intactness of administered immunoglobu 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, lin (e.g., pooled human IgG) is measured by mass spectros 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, copy. For example, the intactness of the administered immu 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or more noglobulins is determined by analyzing a biological sample anti-amyloid B fibril IgG. 60 from the subject, or proteins extracted from the biological In one embodiment, a high titer anti-amyloid B pooled sample, by mass spectroscopy. In some embodiments, the immunoglobulin G composition contains at least 0.04% intactness of the administered immunoglobulins is deter anti-CAPS IgG. In another embodiment, a high titer anti mined by separating proteins present in a biological sample amyloid 3 pooled immunoglobulin G composition contains from the Subject by molecular weight, size, or shape (e.g., by at least 0.08% anti-CAPS IgG. In yet other embodiments, a 65 electrophoresis or size exclusion chromatography) and high titer anti-amyloid 3 pooled immunoglobulin G com determining the size distribution of administered immuno position contains at least 0.04%, 0.05%, 0.06%, 0.07%, globulins in the sample. US 9,556.260 B2 21 22 In one embodiment, the intactness of immunoglobulin non-propellant (e.g., a pump-type inhaler) types of aerosol (e.g., pooled human IgG) in the brain of a Subject following or atomizer devices, particle dispersion devices, nebulizers, intranasal administration is at least 40%. In preferred and pressurized olfactory delivery devices for delivery of embodiments, the intactness of immunoglobulin (e.g., liquid or powder formulations. pooled human IgG) in the brain of a Subject following The term “treatment' or “therapy” generally means intranasal administration is at least 50%, preferably at least obtaining a desired physiologic effect. The effect may be 60%. In certain embodiments, the intactness of immuno prophylactic in terms of completely or partially preventing globulin (e.g., pooled human IgG) in the brain of a subject a disease or condition or symptom thereof and/or may be following intranasal administration is at least 35%, 36%, therapeutic in terms of a partial or complete cure for an 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 10 injury, disease or condition and/or amelioration of an 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, adverse effect attributable to the injury, disease or condition 57%, 58%, 59%, 60%, 61%, or higher. and includes arresting the development or causing regres As used herein, the terms “intranasal administration' and sion of a disease or condition. Treatment can also refer to “nasal administration” refer to administration of a therapeu any delay in onset, amelioration of symptoms, improvement tic composition to the nasal cavity of a Subject such that a 15 in patient Survival, increase in Survival time or rate, therapeutic agent in the composition is delivered directly to improvement in cognitive function, etc. The effect of treat one or more epithelium located in the nose. In certain ment can be compared to an individual or pool of individuals embodiments, intranasal administration is achieved using a not receiving the treatment. liquid preparation (e.g., an aqueous preparation), an aero As used herein, a “therapeutically effective amount or Solized preparation, or a dry powder preparation, each of dose” or “sufficient/effective amount or dose.” refers to a which can be administered via an externally propelled or dose that produces effects for which it is administered. The self-propelled (e.g., via inhalation) non-invasive nasal deliv exact dose will depend on the purpose of the treatment, and ery device, or via a gel, cream, ointment, lotion, or paste will be ascertainable by one skilled in the art using known directly applied to one or more nasal epithelium (e.g., techniques (see, e.g., Lieberman, Pharmaceutical Dosage olfactory epithelium or nasal respiratory epithelium). 25 Forms (vols. 1-3, 1992); Lloyd, The Art, Science and As used herein, the term “nasal epithelium” refers to the Technology of Pharmaceutical Compounding (1999); tissues lining the internal structure of the nasal cavity. The Pickar, Dosage Calculations (1999); and Remington: The term nasal epithelium includes both the nasal respiratory Science and Practice of Pharmacy, 20th Edition, 2003, epithelium, located in the lower two-thirds of the nasal Gennaro, Ed., Lippincott, Williams & Wilkins). cavity in humans, and the olfactory epithelium, located in 30 As used here, the terms “dose' and “dosage' are used the upper third of the nasal cavity of humans. interchangeably and refer to the amount of active ingredient As used herein, the term "olfactory epithelium” refers to given to an individual at each administration. The dose will a specialized epithelial tissue inside the nasal cavity vary depending on a number of factors, including frequency involved in smell. In humans, the olfactory epithelium is of administration; size and tolerance of the individual; located in the upper third of the nasal cavity. 35 severity of the condition: risk of side effects; and the route As used herein, the term "directed administration” refers of administration. One of skill in the art will recognize that to a process of preferentially delivering a therapeutic agent the dose can be modified depending on the above factors or to a first location in a Subject as compared a second location based on therapeutic progress. The term "dosage form or systemic distribution of the agent. For example, in one refers to the particular format of the pharmaceutical, and embodiment, directed administration of a therapeutic agent 40 depends on the route of administration. For example, a results in at least a two-fold increase in the ratio of thera dosage form can be a liquid or dry powder, formulated for peutic agent delivered to a targeted site to therapeutic agent intranasal administration. delivered to a non-targeted site, as compared to the ratio As used herein, a therapeutic composition "consisting following systemic or non-directed administration. In other essentially of a buffering agent and pooled human IgG may embodiments, directed administration of a therapeutic agent 45 also contain residual levels of chemical agents used during results in at least a 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, the manufacturing process, e.g., Surfactants, buffers, salts, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, and Stabilizing agents, as well as chemical agents used to pH 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, the final composition, for example, counter ions contributed 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, by an acid (e.g., hydrochloric acid or acetic acid) or base 750-fold, 1000-fold, or greater increase in the ratio of 50 (e.g., sodium or potassium hydroxide), and/or trace amounts therapeutic agent delivered to a targeted site to therapeutic of contaminating proteins. agent delivered to a non-targeted site, as compared to the As used herein, the term “permeability enhancer refers to ratio following systemic or non-directed administration. In a a component of a therapeutic composition formulated for particular embodiment, directed administration of an agent intranasal administration which promotes the passage of is contrasted to intravenous administration of the agent. For 55 biotherapeutics (e.g., mimetibodies and Fc-fusion polypep example, in one embodiment, the ratio of therapeutic agent tides) through the nasal epithelium. Non-limiting examples present at a targeted site to therapeutic agent present in the of permeability enhancers include membrane fluidizers, blood stream is increased at least two-fold when the agent is tight junction modulators, and medium chain length fatty Subject to directed administration (e.g., by delivery to the acids and salts and esters thereof. Non-limiting examples of brain via intranasal administration), as compared to when 60 medium chain length fatty acids and salts and esters thereof the therapeutic agent is administered intravenously. included mono-, di-, and triglycerides (such as Sodium As used herein, the term “non-invasive nasal delivery caprylate, sodium caprate, glycerides (CAPMUL, GELU device' refers an instrument that is capable of delivering a CIRE 44/14 PEG32 glyceryl laurate EP); lipids; pegylated therapeutic composition (e.g., pooled human IgG) to the peptides; and liposomes. Surfactants and similarly acting nasal cavity without piercing the epithelium of the Subject. 65 compounds can also be used as permeability enhancers. Non-limiting examples of non-invasive nasal delivery Non-limiting examples of Surfactants and similarly acting devices include propellant (e.g., a pressurized inhaler) and compounds include polysorbate-80, phosphatidylcholine, US 9,556.260 B2 23 24 N-methylpiperazine, sodium salicylate, melittin, and palmi there is a direct connection between the olfactory system and toyl carnitine chloride (PCC). Generally, the pooled human the brain. Intranasal administration of IgG (INIG) to treat immunoglobulin G compositions described herein are for neurological diseases is particularly advantageous because mulated for intranasal administration in the absence of the direct connection between the olfactory system and the permeability enhancers. brain obviates delivery concerns associated with the blood As used herein, the term “dry powder composition” refers brain barrier (BBB) and minimizes systemic exposure to the to a lyophilized or spray dried form of a therapeutic pooled drug, thereby minimizing side effects of the drug. Further human IgG formulation. In one embodiment, a dry powder more, IN delivery allows compositions such as powders, composition contains less than 10%, 9%, 8%, 7%, 6%. 5%, granules, solutions, ointments, and creams, thereby obviat 4%, 3%, 2%, 1%, or less residual water content. 10 ing the need for intravenous and intramuscular administra A “control is used herein, refers to a reference, usually a tion. For example, when a drug is administered intranasally, known reference, for comparison to an experimental group. it is transported through the nasal mucosa and along the One of skill in the art will understand which controls are olfactory neural pathway. The drug can be administered valuable in a given situation and be able to analyze data alone or can be combined with a carrier molecule(s) to based on comparisons to control values. Controls are also 15 promote transport through the nasal mucosa and along the valuable for determining the significance of data. For olfactory neural pathway. The drug can also be administered example, if values for a given parameter vary widely in in combination with an absorption enhancer. Absorption controls, variation in test samples will not be considered as enhancers promote the absorption of the drug through the significant. nasal mucosa and along the olfactory neural pathway. Fur Before the present disclosure is described in greater detail, thermore, additional molecules can be added to facilitate it is to be understood that this invention is not limited to drug transport across the olfactory neural pathway. particular embodiments described, as such may, of course, IN administration can also be used to deliver therapeutic vary. It is also to be understood that the terminology used drugs to the brain via the trigeminal pathway. Specifically, herein is for the purpose of describing particular embodi IN administration can be used to deliver IgG via the trigemi ments only, and is not intended to be limiting, since the 25 nal pathway. The olfactory and trigeminal nerves receive scope of the present invention will be limited only by the high concentrations of a drug with IN administration appended claims. because the absorbent respiratory and olfactory pseudoepi Where a range of values is provided, it is understood that thelium are innervated by the trigeminal nerve. These nerves each intervening value, to the tenth of the unit of the lower can then transport the drug into the brain and other con limit unless the context clearly dictates otherwise, between 30 nected structures. For example, the trigeminal nerve the upper and lower limit of that range and any other stated branches directly or indirectly reach the maxillary sinus, or intervening value in that stated range, is encompassed brainstem, hindbrain, cribriform plate, forebrain (e.g., cortex within the invention. The upper and lower limits of these and diencephalon), orofacial structures (e.g., teeth, masseter Smaller ranges may independently be included in the Smaller muscle, and the temporomandibular joint), midbrain, cer ranges and are also encompassed within the invention, 35 ebellum, cervical spinal cord, thoracic spinal cord, and Subject to any specifically excluded limit in the Stated range. lumbar spinal cord. Accordingly, INIG can be carried across Where the stated range includes one or both of the limits, the trigeminal pathway to reach and treat neurological ranges excluding either or both of those included limits are diseases. also included in the invention. In certain embodiments, methods are provided for the Unless defined otherwise, all technical and scientific 40 treatment of CNS disorders by administration of pooled terms used herein have the same meaning as commonly human immunoglobulins to tissue innervated by the olfac understood by one of ordinary skill in the art to which this tory and/or trigeminal nerves. Surprisingly, it was found that invention belongs. Although any methods and materials therapeutically effective amounts of pooled human immu similar or equivalent to those described herein can also be noglobulin are delivered to the CNS when administered used in the practice or testing of the present invention, 45 intranasally. For example, it is shown herein that intranasal representative illustrative methods and materials are now administration of pooled human immunoglobulins is effec described. tive to reduce total amyloid plaque load in a rodent model of It is noted that, as used herein and in the appended claims, Alzheimer's disease. Moreover, by specifically targeting the the singular forms “a,” “an,” and “the include plural nasal epithelium, as opposed to the respiratory system (lung, referents unless the context clearly dictates otherwise. It is 50 pharynx, etc.), systemic exposure of the pooled human further noted that the claims may be drafted to exclude any immunoglobulins is reduced. optional element. As such, this statement is intended to serve Many types of intranasal delivery devices can be used to as antecedent basis for use of such exclusive terminology as practice the methods provided herein. In some embodi “solely,” “only, and the like in connection with the recita ments, the delivery device is an intranasal device for the tion of claim elements, or use of a “negative' limitation. 55 administration of liquids. Non-limiting examples of devices As will be apparent to those of skill in the art upon reading useful for the administration of liquid compositions (e.g., this disclosure, each of the individual embodiments liquid pooled IgG compositions) include vapor devices (e.g., described and illustrated herein has discrete components and vapor inhalers), drop devices (e.g., catheters, single-dose features which may be readily separated from or combined droppers, multi-dose droppers, and unit-dose pipettes), with the features of any of the other several embodiments 60 mechanical spray pump devices (e.g., Squeeze bottles, multi without departing from the scope or spirit of the present dose metered-dose spray pumps, and single/duo-dose spray invention. Any recited method can be carried out in the order pumps), bi-directional spray pumps (e.g., breath-actuated of events recited or in any other order which is logically nasal delivery devices), gas-driven spray systems/atomizers possible. (e.g., single- or multi-dose HFA or nitrogen propellant Administration 65 driven metered-dose inhalers, including traditional and cir Intranasal (IN) administration is an advantageous mode of cumferential velocity inhalers), and electrically powered delivering a drug to the brain because it is non-invasive and nebulizers/atomizers (e.g., pulsation membrane nebulizers, US 9,556.260 B2 25 26 vibrating mechanical nebulizers, and hand-held mechanical The maxillary sinus is in fluid communication with the nebulizers). In some embodiments, the delivery device is an patient's nasal cavity and comprises right and left maxillary intranasal device for the administration of powders or gels. sinuses. Each maxillary sinus communicates with the cor Non-limiting examples of devices useful for the adminis responding nasal passage via the orifice of the maxillary tration of powder compositions (e.g., lyophilized or other sinus. The maximum volume of the maxillary sinus in adults wise dried pooled IgG compositions) include mechanical is approximately 4 to 15 ml, though individual sinuses may powder sprayers (e.g., hand-actuated capsule-based powder comprise Volumes outside of this range. spray devices and hand-actuated powder spray devices, hand The pathway from the nasal passages to the corresponding actuated gel delivery devices), breath-actuated inhalers (e.g., orifice of maxillary sinus, and ultimately to the correspond 10 ing maxillary sinus, allows for a device to be inserted into single- or multi-dose nasal inhalers and capsule-based the nasal passage to the orifice of the maxillary sinus, single- or multi-dose nasal inhalers), and insufllators (e.g., whereupon at least one effective amount or dose of pooled breath-actuated nasal delivery devices). In some embodi human immunoglobulins may be administered and delivered ments, the pooled human immunoglobulins are preferen into the maxillary sinus. The pathway to the maxillary sinus tially administered to the olfactory area, located in the upper 15 is tortuous and requires: traversing the nostril, moving third of the nasal cavity, and particularly to the olfactory through the region between the lower and middle concha, epithelium. Fibers of the olfactory nerve are unmyelinated navigating over and into the semilunar hiatus, traveling axons of olfactory receptor cells, which are located in the Superiorly into the maxillary sinus opening, resisting the superior one-third of the nasal cavity. The olfactory receptor ciliated action of the ostium/tube passing into the maxillary cells are bipolar neurons with swellings covered by hair-like sinus and ultimately moving into the sinus itself. cilia that project into the nasal cavity. At the other end, axons Since the trigeminal nerve passes through the maxillary from these cells collect into aggregates and enter the cranial sinus, the pooled human immunoglobulins in the maxillary cavity at the roof of the nose. Surrounded by a thin tube of sinus after delivery therein will be moved along the trigemi pia, the olfactory nerves cross the Subarachnoid space con nal nerve to structures innervated by the trigeminal nerve. In taining CSF and enter the inferior aspects of the olfactory 25 this fashion, pooled human IgG administered to one or both bulbs. Once the pooled human immunoglobulin is dispensed of the maxillary sinus is delivered to the brain via the into the nasal cavity, the immunoglobulin can undergo trigeminal nerve. transport through the nasal mucosa and into the olfactory In one embodiment, the pooled human IgG compositions bulb and interconnected areas of the brain, such as the provided herein for the treatment of a CNS disorder (e.g., hippocampal formation, amygdaloid nuclei, nucleus basalis 30 Alzheimer's disease) are intranasally administered as a of Meynert, locus ceruleus, the brain stem, and the like (e.g., liquid preparation, e.g., an aqueous based preparation. For Johnson et al., Molecular Pharmaceutics (2010) 7(3):884 example, in one embodiment, nasal drops are instilled in the 893). nasal cavity by tilting the head back sufficiently and apply In certain embodiments, pooled human immunoglobulin the drops into the nares. In another embodiment, the drops is administered to tissue innervated by the trigeminal nerve. 35 are Snorted up the nose. In another embodiment, nasal drops The trigeminal nerve innervates tissues of a mammals (e.g., are applied with an applicator or tube onto the upper third of human) head including skin of the face and Scalp, oral the nasal mucosa. In another embodiment, nasal drops are tissues, and tissues Surrounding the eye. The trigeminal applied with an applicator or tube into one or both of nerve has three major branches, the ophthalmic nerve, the maxillary sinus of the subject. In another embodiment, the maxillary nerve, and the mandibular nerve. In some embodi 40 liquid preparation may be placed into an appropriate device ments, the methods provided herein include targeted admin so that it may be aerosolized for inhalation through the nasal istration of pooled human immunoglobulinto one or more of cavity. For example, in one embodiment, the therapeutic these trigeminal branches, i.e. the trigeminal pathway. In agent is placed into a plastic bottle atomizer. In a specific some embodiments, the methods provided herein include embodiment, the atomizer is advantageously configured to targeted administration of pooled human immunoglobulin to 45 allow a substantial amount of the spray to be directed to the the maxillary sinus, thereby reaching the brainstem, hind upper one-third region or portion of the nasal cavity (e.g., brain, cribriform plate, forebrain (e.g., cortex and dien the olfactory epithelium). In another embodiment, the liquid cephalon), midbrain, cerebellum, cervical spinal cord, tho preparation is aerosolized and applied via an inhaler, such as racic spinal cord, and lumbar spinal cord through the a metered-dose inhaler (for example, see, U.S. Pat. No. trigeminal pathway. In certain embodiments, methods pro 50 6,715.485). In a specific embodiment, the inhaler is advan vided herein include targeted administration of pooled tageously configured to allow a Substantial amount of the human immunoglobulin for treatment of a disorder of the aerosol to be directed to the upper one-third region or CNS (e.g., Alzheimer's disease). portion of the nasal cavity (e.g., the olfactory epithelium). In In some embodiments, the pooled human immunoglobu certain embodiments, a Substantial amount of the pooled lin is administered to nasal tissues innervated by the trigemi 55 human immunoglobulin refers to at least 25%, 30%, 35%, nal nerve, for example, to nasal tissues including the sinuses, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, the inferior two-thirds of the nasal cavity and the nasal 90%. 95%, or 100% of the composition, which is adminis septum. In certain embodiments, the pooled human immu tered to the upper one-third region of the nasal cavity (e.g., noglobulin is targeted to the inferior two-thirds of the nasal administered to the upper one-third of the nasal epithelium). cavity and/or the . 60 In one embodiment, the pooled human IgG compositions In some embodiments, the pooled human immunoglobu provided herein for the treatment of a CNS disorder (e.g., lin is administered to one or both maxillary sinus of the Alzheimer's disease) are intranasally administered as a dry individual. Methods and devices for administration to the powder. Dry powder nasal delivery devices are well known maxillary sinus are known in the art, for example, see United in the art, for example, see PCT publication No. WO States Patent Application Publication Number 2011/ 65 1996/222802. In one embodiment, following intranasal 0151393, the contents of which are hereby incorporated by administration, pooled human IgG is absorbed across the reference in their entirety for all purposes. olfactory epithelium, which is found in the upper third of the US 9,556.260 B2 27 28 nasal cavity. In another embodiment, following intranasal ery device delivers a dry powder composition of pooled administration, pooled human IgG is absorbed across the human IgG directly to a nasal epithelium of the Subject. In nasal respiratory epithelium, which is innervated with a more specific embodiment, the non-invasive intranasal trigeminal nerves, in the lower two-thirds of the nasal cavity. delivery device delivers a dry powder composition of pooled The trigeminal nerves also innervate the conjunctive, oral human IgG directly to the olfactory epithelium of the mucosa, and certain areas of the dermis of the face and head, Subject. and absorption after intranasal administration of the IgG In another embodiment, the non-invasive intranasal deliv from these regions may also occur. In other embodiments, ery device delivers a sustained release or controlled release following intranasal administration, pooled human IgG is composition of pooled human IgG composition to the nasal absorbed across the maxillary sinus epithelium. In yet other 10 cavity of a Subject. In a specific embodiment the Sustained embodiments, pooled human IgG may be absorbed across release composition comprises a dry powder composition of more than one of these nasal epitheliums and Subsequently pooled human IgG. In some embodiments, the Sustained delivered to the brain of the subject. release composition is a gel, paste, hydrogel, cream, lotion, Although administration is referred to herein as a single film, or similar form that coats at least a portion of the nasal event that may occur according to Some regular or irregular 15 epithelium (e.g., all or a portion of the olfactory epithelium, frequency of the course of a treatment, a single administra all or a portion of a nasal epithelium associated with tion even may include multiple administrations. In this trigeminal nerve endings, all or a portion of the upper third regard, a single dosage of pooled human IgG may be of the nasal epithelium, all or a portion of the lower third of partitioned into two or more physical compositions for the nasal epithelium, or all or a portion of the nasal maxillary administration. For example, a 200 mg dose of pooled epithelium. human IgG in a liquid composition formulated at 200 g/L In one embodiment, the intranasal device is a single-use, IgG may be administered to a 50 kg Subject (4 mg/kg IgG) disposable device. In another embodiment, the intranasal in four drops having a Volume of 250 uL each. Likewise, a device is a multi- or repeat-use device. In certain embodi dry powder composition containing a single dosage of ments, the single-use or multi-use device is pre-metered. In pooled human IgG may be administered, for example, in two 25 a specific embodiment, the single-use or multi-use device is or more distinct puffs. In some embodiments, pooled human pre-filled. In certain embodiments, the multi- or repeat-use IgG is administered in one or more puffs or sprays into each device is refillable. In certain embodiments, the device is nare of the individual (e.g., one or more puff into the right refilled by inserting a pooled human IgG composition into a nare and one or more puffs into the left nare). chamber of the device. In other embodiments, a chamber of In certain embodiments, the methods described herein for 30 the multi- or repeat-use device designed to hold the pooled treating a CNS disorder include intranasal administration of human IgG composition is replaced with a new, pre-filled pooled human IgG via a non-invasive intranasal delivery chamber. device. In one embodiment, the non-invasive intranasal In certain embodiments, the pooled human immunoglobu delivery device is a non-propellant type aerosol or atomizer lin compositions are administered by a pressurized nasal device, a propellant type aerosol or atomizer device, a 35 delivery (PND) device. In one embodiment, the PND device non-propellant pump-type device, a particle dispersion can be used to deliver a liquid IgG composition to the nasal device, a nebulizer device, or a pressurized olfactory deliv cavity. In one embodiment, the PND device can be used to ery device. deliver a powder IgG composition to the nasal cavity. In one In one embodiment the non-invasive intranasal delivery embodiment, the PND device administers an IgG composi device delivers a liquid drop of a pooled human IgG 40 tion into one nostril. In one embodiment, the Impel device composition to the nasal cavity of a subject. In a particular administers an IgG composition into both nostrils. embodiment, the non-invasive intranasal delivery device In some embodiments, the PND device is configured to delivers a liquid drop of pooled human IgG directly to a deliver the liquid or powder IgG compositions to a particular nasal epithelium of the Subject. In a more specific embodi epithelium, location, and/or structure of the nasal cavity. For ment, the non-invasive intranasal delivery device delivers a 45 example, in one embodiment, the PND device is configured liquid drop of pooled human IgG directly to the olfactory to deliver the IgG composition to the upper nasal cavity. In epithelium of the subject. In one embodiment, the liquid one embodiment, the PND device is configured to deliver drop is administered by tilting the head of the subject back the IgG composition to the olfactory epithelium of the nasal and administering the drop into a nare of the Subject. In cavity. In one embodiment, the PND device is configured to another embodiment, the liquid drop is administered by 50 deliver the IgG composition to the lower two thirds of the inserting the tip of a non-invasive intranasal delivery device nasal epithelium. In one embodiment, the PND device is into a nare of the Subject and squirting or spraying the drop configured to deliver the IgG composition to a nasal epithe into the nasal cavity of the subject. lium associated with trigeminal nerve endings. In one In another embodiment, the non-invasive intranasal deliv embodiment, the PND device is configured to deliver the ery device delivers a liquid or a powder aerosol of a pooled 55 IgG composition to the nasal maxillary sinus epithelium. human IgG composition to the nasal cavity of a subject. In Methods for configuring pressurized delivery devices to a particular embodiment, the non-invasive intranasal deliv achieve a particular delivery profile are known in the field. ery device delivers a liquid or a powder aerosol of pooled For example, in one embodiment, a pressurized nasal deliv human IgG directly to a nasal epithelium of the Subject. In ery device is configured to produce a stream, spray, puff, a more specific embodiment, the non-invasive intranasal 60 etc., have a particular characteristic. For example, in one delivery device delivers a liquid or a powder aerosol of embodiment, to achieve administration to the upper third of pooled human IgG directly to the olfactory epithelium of the the nasal epithelium, the device is configured to produce a Subject. strong, focused stream, spray, puff, etc. In one embodiment, In another embodiment, the non-invasive intranasal deliv the strong focused spray is created by imparting circumfer ery device delivers a dry powder composition of pooled 65 ential and/or axial velocity onto the stream of the therapeutic human IgG composition to the nasal cavity of a subject. In composition (e.g., pooled human IgG) being administered a particular embodiment, the non-invasive intranasal deliv into the nose. In another embodiment, to achieve adminis US 9,556.260 B2 29 30 tration to a greater portion of the nasal epithelium (e.g., the In one embodiment, an intranasal device described herein entire or the lower two thirds of the nasal epithelium), the can deliver 80%-90% of the metered IgG dose to the device is configured to produce a diffuse and/or weaker olfactory region. In one embodiment, an intranasal device stream, spray, puff, etc. In some embodiments, the tip of the described herein can deliver 60%-80% of the metered IgG delivery device is configured to physically direct the stream, 5 dose to the olfactory region. spray, puff, etc., to the desired intranasal location when inserted into the subject’s nare. For example, a kink or bend In certain embodiments, the pooled human immunoglobu may be introduced into the tip of the delivery device to lin compositions are administered by an intranasal device "point the stream, spray, puff, etc., at a targeted epithelium. described above in one or more doses. In one embodiment In some embodiments, the delivery pattern of the device is the more than one dose is administer by the intranasal device adjustable, such that the device can be differentially config 10 in alternating nostrils. In one embodiment, the more than ured to target the therapeutic agent (e.g., pooled human IgG) one does is administered by the intranasal device at different to a particular epithelium, structure, or location within the time points throughout the day. In certain embodiments the nose. In certain embodiments, the pooled human immuno more than one dose is two, three, four, five, six, seven, eight, globulin compositions are administered by a breath-powered nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, technology device. In certain embodiments, the breath 15 powered technology provides positive pressure during seventeen, eighteen, nineteen, or twenty or more doses. In administration. In certain embodiments, the positive pres certain embodiments the more than one dose is administered Sure expands narrow nasal passages. In certain embodi by the intranasal device one, two, three, four, five, six, seven, ments, the expansion of the nasal passages allows reliable eight, nine, or ten or more time points throughout the day. delivery of liquid or powderpooled human immunoglobulin In certain embodiments, the pooled human immunoglobu compositions described herein to the CNS. In some embodi lin compositions are administered by an intranasal device ments, exhalation into the device propels the therapeutic described above in an initial dose or set of doses followed by (e.g., pooled human IgG) into the nose, while at the same repeat maintenance doses. In certain embodiments the initial time closing the soft-palette, thereby reducing deposition of dose is one, two, three, four, five, six, seven, eight, nine, ten, the therapeutic into the throat and/or lungs. In one embodi 25 eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, ment, the breath-powered technology device administers an eighteen, nineteen, or twenty or more doses. IgG composition described herein into one nostril. In one In another embodiment, a gel, cream, ointment, lotion, or embodiment, the breath-powered technology device admin paste containing pooled human IgG is applied onto the nasal isters an IgG composition described herein into two nostrils. epithelium, for example, by use of an application stick or Non-limiting examples of commercial intranasal delivery 30 Swab. In a particular embodiment, a gel, cream, ointment, devices include the EQUADEL(R) nasal spray pump (Aptar lotion, or paste containing pooled human IgG is applied Pharma), the Solovent dry powder device (BD Technolo directly onto a nasal epithelium of the subject. In a more gies), the Unidose nasal drug delivery device (Consort specific embodiment, a gel, cream, ointment, lotion, or paste Medical PLC), the NasoNeb(R) Nasal Nebulizer (MedInvent, containing pooled human IgG is applied directly onto the LLC), the Veriloser R nasal delivery device (Mystic Phar 35 olfactory epithelium of the subject. maceuticals), the VRX2TM nasal delivery device (Mystic In certain embodiments, a substantial fraction of the Pharmaceuticals), the DirectHalerTM Nasal device (Direct therapeutic agent present in the composition is delivered Haler A/S), the TriViarTM single-use unit-dose dry powder directly to one or more nasal epithelium. In certain embodi inhaler (Trimel Pharmaceuticals), the SinuStarTM Aerosol ments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, Delivery System (Pari USA), the Aero Pump (Aero Pump 40 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the GmbH), the Fit-LizerTM nasal delivery device (Shin Nippon therapeutic agent present in the composition is delivered Biomedical Laboratories), the LMA MAD NasalTM device directly to a nasal epithelium. In a specific embodiment, a (LMA North America, Inc.), the Compleo intranasal bioad Substantial fraction of the therapeutic agent present in the hesive gel delivery system (Trimel Pharmaceuticals), composition is delivered directly to the olfactory epithelium. Impel's Pressurized Olfactory Delivery (POD) device (Im 45 In a more specific embodiment, at least 25%, 30%, 35%, pel Neuropharma), the ViaNaseTM electronic atomizer 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, (Kurve Technology, Inc.), the OptiNose powder delivery 90%. 95%, or 100% of the therapeutic agent present in the device (OptiNose US Inc.), and the Optinose liquid delivery composition is delivered directly to the olfactory epithelium. device (OptiNose US Inc.) In another specific embodiment, a substantial fraction of the In one embodiment, an intranasal device described herein 50 therapeutic agent present in the composition is delivered can deliver 10%-20% of the metered IgG dose to the directly to nasal epithelium innervated with trigeminal olfactory region. In one embodiment, an intranasal device nerves. In a more specific embodiment, at least 25%, 30%, described herein can deliver 20%-30% of the metered IgG 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, dose to the olfactory region. In one embodiment, an intra 85%, 90%, 95%, or 100% of the therapeutic agent present in nasal device described herein can deliver 5%-20% of the 55 the composition is delivered directly to nasal epithelium metered IgG dose to the olfactory region. In one embodi innervated with trigeminal nerves. ment, an intranasal device described herein can deliver In some embodiments, pooled human IgG can be admin 30%-40% of the metered IgG dose to the olfactory region. istered to a Subject as a combination therapy with another In one embodiment, an intranasal device described herein treatment, e.g., another treatment for a disorder of the central can deliver 40%-50% of the metered IgG dose to the 60 nervous system (e.g., Alzheimer's disease, age-related olfactory region. In one embodiment, an intranasal device dementia, Parkinson's disease, or multiple Sclerosis). For described herein can deliver 60%–70% of the metered IgG example, the combination therapy can include administering dose to the olfactory region. In one embodiment, an intra to the Subject (e.g., a human patient) one or more additional nasal device described herein can deliver 60%-80% of the agents that provide a therapeutic benefit to the subject who metered IgG dose to the olfactory region. In one embodi 65 has, or is at risk of developing, a disorder of the central ment, an intranasal device described herein can deliver nervous system, e.g., Alzheimer's disease. In some embodi 70%–80% of the metered IgG dose to the olfactory region. ments, the pooled human IgG and the one or more additional US 9,556.260 B2 31 32 agents are administered at the same time. In other embodi 84-89); Perlmutter, et al. 1999 (The Lancet, 354:1153-1158); ments, the pooled human IgG is administered first in time Snider et al. 2003 (J. of Child and Adolescent Psychophar and the one or more additional agents are administered macology, 13(supp 1): S81-S88). In these trials, subjects are second in time. In some embodiments, the one or more administered between 0.4 g/kg body weight and 2.0 g/kg additional agents are administered first in time and the body weight IVIG per dosage. Specifically, the treatment pooled human IgG is administered second in time. regimes of CNS disorders with IVIG range from 0.4 g/kg The pooled human IgG can replace or augment a previ body weight IVIG administered once daily for 5 consecutive ously or currently administered therapy. For example, upon days to 2.0 g/kg body weight IVIG administered once daily treating with pooled human IgG, administration of the one for 2 consecutive days. There are several variations of these or more additional agents can cease or diminish, e.g., be 10 IVIG treatment regimes. For example, IVIG treatment administered at lower levels. In other embodiments, admin regimes may be 1.0 g/kg body weight IVIG administered istration of the previous therapy is maintained. In some twice a day (total 2.0 g/kg body weight IVIG per day). The embodiments, a previous therapy will be maintained until initial 2 to 5 day IVIG dosages can also be followed with the level of polyclonal IgG reaches a level sufficient to maintenance doses ranging from 0.4 g/kg to 0.5 g/kg body provide a therapeutic effect. The two therapies can be 15 weight IVIG. Due to the limited supply of pooled human administered in combination. IgG, and high cost associated therewith, large-scale imple In one embodiment, a human receiving a first therapy for mentation of these treatments may prove problematic if they a disorder of the central nervous system, e.g., Alzheimer's are approved by major regulatory bodies. disease, who is then treated with pooled human IgG, con Typical intravenous dosing of IgG in human Alzheimer's tinues to receive the first therapy at the same or a reduced trials ranges from 200 mg/kg to 400 mg/kg every two weeks. amount. In another embodiment, treatment with the first Advantageously, the inventors have found that levels of therapy overlaps for a time with treatment with pooled pooled human IgG seen in the brain after intravenous human IgG, but treatment with the first therapy is subse administration can also be achieved by intranasal adminis quently halted. tration. For example, it is shown in Example 3 that admin In a particular embodiment, pooled human IgG may be 25 istration of pooled human IgG (0.02 g/kg IgG) intranasally administered in combination with a treatment for an age as drops (IN1) or a liquid spray delivered directly to the related dementia, e.g., Alzheimer's disease. In certain olfactory epithelium (IN3) results in substantially the same embodiments, the treatment for an age-related dementia amount of IgG being delivered to the right and left hemi co-administered with pooled human IgG is administration of spheres of the brain as for intravenous administration of a cholinesterase inhibitor (e.g., ARICEPT (donepezil), 30 pooled human IgG (0.02 g/kg IgG, compare corrected AUC EXELON (rivastigmine), RAZADYNE (galantamine), or values for right and left hemisphere IgG delivery in Table COGNEX (tacrine), or an inhibitor of the NMDA-type 69, Table 71, and Table 72). Significantly, intranasal admin glutamate receptor (e.g., memantine). istration of IgG liquid drops at concentrations ten-fold lower In further embodiments the second therapy is levodopa (0.002 g/kg IgG) also resulted in the delivery of intact IgG (L-DOPA). The second therapy can also be a dopamine 35 to the cerebral cortex (see, Table 70). Any reduction in the agonist. Non-limiting examples of dopamine agonists amount of pooled human IgG required for administration is include bromocriptine, pergolide, pramipexole, ropinirole, significant because of the limited Supply of pooled human piribedil, cabergoline, apomorphine and lisuride. The sec IgG and the high cost associated therewith. ond therapy can be a MAO-B inhibitor. Non-limiting Accordingly, in certain embodiments, the methods for examples of MAO-B inhibitors are selegiline and rasgiline. 40 treating a CNS disorder provided herein include intranasally Addition second therapies can include amantaine, anticho administering from about 0.05 mg of pooled human IgG per linergic compositions, clozapine, modafinil, and non-steroi kg body weight (mg/kg IgG) to about 500 mg/kg IgG in a dal anti-inflammatory drugs. single dosage. In further embodiments the second therapy is CAMPATH In certain embodiments, the methods for treating a CNS (alemtuzumab), ZENAPX (daclizumab), rituximab, diru 45 disorder provided herein include intranasally administering cotide, BHT-3009, cladribine, dimethyl fumarate, estriol, a low dose of pooled human IgG. In one embodiment, a low laquinimod, pegylated interferon-B-1a, minocycline, statins, dose of pooled human IgG is from about 0.05 mg/kg IgG to temsirolimus, teriflunomide, and low dose naltexone. about 10 mg/kg IgG. In specific embodiments, a low dose of In certain embodiments the second therapy is psycho pooled human IgG is about 0.05 mg/kg, 0.06 mg/kg, 0.07 therapy. Non-limiting examples of psychotherapy are psy 50 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.10 mg/kg, 0.15 mg/kg, chosocial intervention, behavioral intervention, reminis 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 cence therapy, validation therapy, Supportive psychotherapy, mg/kg, 0.45 mg/kg, 0.50 mg/kg, 0.55 mg/kg, 0.60 mg/kg, sensory integration, simulated presence therapy, cognitive 0.65 mg/kg, 0.70 mg/kg, 0.75 mg/kg, 0.80 mg/kg, 0.85 retraining, and stimulation-oriented therapies Such as art, mg/kg, 0.90 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 music, pet, exercise, and recreational activities. 55 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 Furthermore, two or more second therapies can be com mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 bined with therapeutic intranasal IgG. For example, thera mg/kg, or 10.0 mg/kg IgG. In yet other embodiments, a low peutic intranasal IgG can be combined with memantine and dose of pooled human IgG is from 0.1 mg/kg to 5 mg/kg, 0.5 donepezil. mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, 2 mg/kg to 5 mg/kg, Dosing 60 0.5 mg/kg to 10 mg/kg, 1 mg/kg to 10 mg/kg, 2 mg/kg to 10 The use of intravenous immunoglobulin G (IVIG) for the mg/kg, 1 mg/kg to 8 mg/kg, 2 mg/kg to 8 mg/kg, 3 mg/kg treatment of disorders of the central nervous system (CNS) to 8 mg/kg, 4 mg/kg to 8 mg/kg, 5 mg/kg to 8 mg/kg. 1 is currently under investigation (Awad et al. 2011 (Current mg/kg to 6 mg/kg, 2 mg/kg to 6 mg/kg, 3 mg/kg to 6 mg/kg, Neuropharmacology, 9:417428); Pohl et al. 2012 (Current 4 mg/kg to 6 mg/kg, 5 mg/kg to 6 mg/kg, 1 mg/kg to 4 Treatment Options in Neurology, 14:264-275); Krause et al. 65 mg/kg, 2 mg/kg to 4 mg/kg, or 3 mg/kg to 4 mg/kg IgG. 2012 (European J. of Paediatric Neurology, 16:206-208); In certain embodiments, the methods for treating a CNS Elovaara et al. 2011 (Clinical Neuropharmacology, 34(2): disorder provided herein include intranasally administering US 9,556.260 B2 33 34 a medium dose of pooled human IgG. In one embodiment, monthly. For example, a subject diagnosed with a CNS a medium dose of pooled human IgG is from about 10 mg/kg disorder in an early stage of progression may require only a IgG to about 100 mg/kg IgG. In specific embodiments, a low dosage and/or low dosage frequency, while a subject medium dose of pooled human IgG is about 10 mg/kg, 11 diagnosed with a CNS disorder in a late stage of progression mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 5 may require a high dose and/or high dosage frequency. In yet 17 mg/kg, 18 mg/kg, 19 mg/kg. 20 mg/kg, 21 mg/kg, 22 another embodiment, a subject having a high likelihood of mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, developing a CNS disorder may also be prescribed a low 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, dose and/or low dosing frequency as a prophylactic treat 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 10 ment or to delay onset of symptoms associated with a CNS mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg, disorder. For example, a subject with a familial history of an 50 mg/kg, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100 mg/kg age-related dementia (e.g., Alzheimer's disease) may be IgG. In yet other embodiments, a medium dose of pooled intranasally administered pooled human IgG at a low dosage human IgG is from 10 mg/kg to 100 mg/kg, 25 mg/kg to 100 and/or low frequency to delay the onset of symptoms mg/kg, 50 mg/kg to 100 mg/kg, 75 mg/kg to 100 mg/kg, 10 15 associated with the age-related dementia. A skilled physician mg/kg to 75 mg/kg, 25 mg/kg to 75 mg/kg, 50 mg/kg to 75 will readily be able to determine an appropriate dosage and mg/kg, 10 mg/kg to 50 mg/kg, 25 mg/kg to 50 mg/kg, or 10 dosing frequency for a Subject diagnosed with or having a mg/kg to 25 mg/kg IgG. high likelihood of developing a CNS disorder. In some embodiments, the methods for treating a CNS In one embodiment, where the progression of a particular disorder provided herein include intranasally administering 20 CNS disorder in a subject requires frequent dosing, the a high dose of pooled human IgG. In one embodiment, a methods provided herein for treating a disorder of the central high dose of pooled human IgG is from about 100 mg/kg nervous system include administering a composition com IgG to about 400 mg/kg IgG. In specific embodiments, a prising pooled human immunoglobulin G (IgG) to the high dose of pooled human IgG is about 100 mg/kg, 110, Subject at least once a week. In other embodiments, the 120 mg/kg, 130 mg/kg, 140 mg/kg, 150 mg/kg, 175 mg/kg, 25 method includes administering a composition comprising 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, pooled human immunoglobulin G (IgG) to the Subject at 325 mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, or higher. In least two, three, four, five, or six times a week. In yet another yet other embodiments, a high dose of pooled human IgG is embodiment, the method includes administering a compo from 100 mg/kg to 400 mg/kg, 150 mg/kg to 400 mg/kg, 200 sition comprising pooled human immunoglobulin G (IgG) to mg/kg to 400 mg/kg, 250 mg/kg to 400 mg/kg, 300 mg/kg 30 the subject at least once daily. In other embodiments, the to 400 mg/kg, 350 mg/kg to 400 mg/kg, 100 mg/kg to 300 method includes administering a composition comprising mg/kg, 150 mg/kg to 300 mg/kg, 200 mg/kg to 300 mg/kg, pooled human immunoglobulin G (IgG) to the subject at 250 mg/kg to 300 mg/kg, 100 mg/kg to 200 mg/kg, 150 least two, three, four, five, or more times daily. In a specific mg/kg to 200 mg/kg, or 100 mg/kg to 150 mg/kg IgG. embodiment, the CNS disorder is an age-related dementia, In some embodiments, pooled human IgG is administered 35 Parkinson's disease, or multiple Sclerosis. In a more specific at a set dosage, regardless of the weight of the Subject. embodiment, the CNS disorder is Alzheimer's disease. Without being bound by theory, unlike intravenous admin In another embodiment, where the progression of a par istration, the final concentration of IgG in the brain should ticular CNS disorder in a subject requires less frequent be independent of total body weight when administered dosing, the methods provided herein for treating a disorder intranasally since the therapeutic will travel directly from 40 of the central nervous system include administering a com the nose to the brain. Accordingly, a standard dose of position comprising pooled human immunoglobulin G (IgG) intranasal pooled human IgG, which is independent of body to the Subject at least once a month. In other embodiments, weight, may simplify the process of dosing individual Sub the method includes administering a composition compris jects. ing pooled human immunoglobulin G (IgG) to the Subject at Accordingly, in one embodiment, the methods described 45 least two, three, four, five, six, or more times a month. In yet herein include intranasal administration of a fixed dose of another embodiment, the method includes administering a pooled human IgG of from about 50 mg to about 10 g. In composition comprising pooled human immunoglobulin G some embodiments, the fixed dose of IgG is about 50 mg, 75 (IgG) to the subject at least once daily. In other embodi mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 ments, the method includes administering a composition mg, 275 mg. 300 mg, 350 mg. 400 mg. 450 mg, 500 mg, 550 50 comprising pooled human immunoglobulin G (IgG) to the mg, 600 mg. 650 mg, 700 mg, 750 mg. 800 mg. 850 mg,900 Subject at least two, three, four, five, or more times daily. In mg, 950 mg, 1.0 g, 1.25 g, 1.5 g, 1.75 g, 2.0 g, 2.5 g., 3.0 g, a specific embodiment, the CNS disorder is an age-related 3.5 g. 4.0 g, 4.5 g., 5.0 g, 5.5 g. 6.0 g. 6.5g, 7.0 g, 7.5g, 8.0 dementia, Parkinson's disease, or multiple Sclerosis. In a g, 8.5 g., 9.0 g, 9.5 g. 10.0 g, or more IgG. In other more specific embodiment, the CNS disorder is Alzheimer's embodiments, the methods described herein include intra- 55 disease. nasal administration of from 50 mg to 5 g, 100 mg to 5 g, In certain embodiments, the composition can be admin 250 mg to 5 g, 500 mg to 5 g, 750 mg to 5g, 1 g to 5g, 2.5 istered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, g to 5 g, 50 mg to 2.5 g., 100 mg to 2.5g, 250 mg to 2.5 g. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 times 500 mg to 2.5g, 750 mg to 2.5 g, 1 g to 2.5 g. 50 mg to 1 a month. The composition can be administered between g, 100 mg to 1 g, 250 mg to 1 g, 500 mg to 1 g, 750 mg to 60 equally spaced days of the month, for example, on the 1 and 1 g, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg. the 15" of each month. Alternatively, the composition can be 50 mg to 250 mg, 100 mg to 250 mg. or 50 mg to 100 mg administered in block dosing at the beginning, end, or pooled human IgG. middle of the month. For example, the composition can be Depending upon the CNS disorder being treated and the administered only on the 1, 1'-2', 1'-3", 1'-4". 1'-5", progression of the disorder in the Subject, the pooled human 65 1'-6", 6", or 1'-7" days of the month. Similar dosing IgG compositions described herein are intranasally admin schemes can be administered toward the middle or end of the istered to a subject anywhere from several times daily to month. US 9,556.260 B2 35 36 In certain embodiments the dosing can change between In certain embodiments, the methods provided herein for dosing days. For example, on the first day of dosing a subject the treatment of a CNS disorder include intranasally admin can receive 10 mg/kg IgG and on the second day of dosing istering from 0.05 mg/kg to 50 mg/kg pooled human immu the Subject can receive 20 mg/kg IgG. Similarly, a subject noglobulin to a subject in need thereof daily. In other who is administered two or more doses per day of intranasal embodiments, the methods provided herein for the treatment IgG can receive two different doses. For example, the first of a CNS disorder include intranasally administering pooled dose of the day can be 10 mg/kg IgG and the second dose human IgG in a dosage/frequency combination selected of the day can be 5 mg/kg IgG. from variations 1 to 816 found in Table 1 and Table 2. TABLE 1. Exemplary combinations of dosage and frequency for methods of treating a CNS disorder by intranasal administration of pooled human IgG. One Two Three Four One Two Three Every Time Times Times Times Time Times Times Other Monthly Monthly Monthly Monthly Weekly Weekly Weekly Day

O.OS War. 1 War. S2 War. 103 War. 154 War. 205 War. 256 War. 307 War. 358 mg/kg O.1 War. 2 War. S3 War. 104 War. 155 War. 206 War. 257 War. 308 War. 359 mg/kg O.25 War. 3 War. 54 War. 105 War. 156 War. 207 War. 258 War. 309 War. 360 mg/kg O.S War. 4 War. SS War. 106 War. 157 War. 208 War. 259 War. 310 War. 361 mg/kg 0.75 War. 5 War. 56 War. 107 War. 158 War. 209 War. 260 War. 311 War. 362 mg/kg 1.O War. 6 War. S7 War. 108 War. 159 War. 210 War. 261 War. 312 War. 363 mg/kg 1.5 War. 7 War. S8 War. 109 War. 16O War. 211 War. 262 War. 313 War. 364 mg/kg 2.0 War. 8 War. S9 War. 110 War. 161 War. 212 War. 263 War. 314 War. 365 mg/kg 2.5 War. 9 War. 60 War. 111 War. 162 War. 213 War. 264 War. 315 War. 366 mg/kg 3.0 War. 10 War. 61 War. 112 War. 163 War. 214 War. 265 Var. 316 War. 367 mg/kg 3.5 War. 11 War. 62 War. 113 War. 164 War. 215 War. 266 War. 317 War. 368 mg/kg 4.0 War. 12 War. 63 War. 114 War. 165 War. 216 War. 267 War. 318 War. 369 mg/kg 4.5 War. 13 War. 64 War. 115 War. 166 War. 217 War. 268 War. 319 War. 370 mg/kg S.O War. 14 War. 65 War. 116 War. 167 War. 218 War. 269 War. 32O War. 371 mg/kg 6.O War. 15 War. 66 War. 117 War. 168 War. 219 War. 270 War. 321 War.372 mg/kg 7.0 War. 16 War. 67 War. 118 War. 169 War. 22O War. 271 War. 322 War. 373 mg/kg 8.0 War. 17 War. 68 War. 119 War. 17O War. 221 War. 272 War. 323 War. 374 mg/kg 9.0 War. 18 War. 69 War. 120 War. 171 War. 222 War. 273 War. 324 War.375 mg/kg O War. 19 War. 70 War. 121 War. 172 War. 223 War. 274 War. 325 War. 376 mg/kg 1 War. 20 War. 71 War. 122 War. 173 War. 224 War. 275 War. 326 War.377 mg/kg 2 War. 21 War. 72 War. 123 War. 174 War. 225 War. 276 War. 327 War. 378 mg/kg 3 War. 22 War. 73 War. 124 War. 175 War. 226 War. 277 War. 328 War. 379 mg/kg 4 War. 23 War. 74 War. 125 War. 176 War. 227 War. 278 War. 329 War. 380 mg/kg 5 War. 24 War. 75 War. 126 War. 177 War. 228 War. 279 War. 330 War. 381 mg/kg 6 War. 25 War. 76 War. 127 War. 178 War. 229 War. 280 War. 331 War. 382 mg/kg 7 War. 26 War. 77 War. 128 War. 179 War. 230 War. 281 War. 332 War. 383 mg/kg 8 War. 27 War. 78 War. 129 War. 180 War. 231 War. 282 War. 333 War. 384 mg/kg 9 War. 28 War. 79 War. 130 War. 181 War. 232 War. 283 War. 334 War. 385 mg/kg 2O War. 29 War. 8O War. 131 War. 182 War. 233 War. 284 War. 335 War. 386 mg/kg 22.5 War. 30 War. 81 War. 132 War. 183 War. 234 War. 285 War. 336 War. 387 mg/kg 25 War. 31 War. 82 War. 133 War. 184 War. 235 War. 286 War. 337 War. 388 mg/kg US 9,556.260 B2 37 38 TABLE 1-continued Exemplary combinations of dosage and frequency for methods of treating a CNS disorder by intranasal administration of pooled human IgG. One Two Three Four One Two Three Every Time Times Times Times Time Times Times Other Monthly Monthly Monthly Monthly Weekly Weekly Weekly Day 27.5 War. 32 War. 83 War. 134 War. 185 War. 236 War. 287 War. 338 War. 389 mg/kg 30 War. 33 War. 84 War. 135 War. 186 War. 237 War. 288 War. 339 War. 390 mg/kg 32.5 War. 34 War. 85 War. 136 War. 187 War. 238 War. 289 War. 340 War. 391 mg/kg 35 War. 35 War. 86 War. 137 War. 188 War. 239 War. 290 War. 341 War. 392 mg/kg 37.5 War. 36 War. 87 War. 138 War. 189 War. 240 War. 291 War. 342 War. 393 mg/kg 40 War. 37 War. 88 War. 139 War. 190 War. 241 War. 292 War. 343 War. 394 mg/kg 45 War. 38 War. 89 War. 140 War. 191 War. 242 War. 293 War. 344 War. 395 mg/kg 50 War. 39 War. 90 War. 141 War. 192 War. 243 War. 294 War. 345 War. 396 mg/kg OS-40 War. 40 War. 91 War. 142 War. 193 War. 244 War. 295 War. 346 War. 397 mg/kg OS-30 War. 41 War. 92 War. 143 War. 194 War. 245 War. 296 War. 347 War. 398 mg/kg OS-20 War. 42 War. 93 War. 144 War. 195 War. 246 War. 297 War. 348 War. 399 mg/kg OS-20 War. 43 War. 94 War. 145 War. 196 War. 247 War. 298 War. 349 War. 400 mg/kg O5-10 War. 44 War. 95 War. 146 War. 197 War. 248 War. 299 War. 350 War. 401 mg/kg O.S.-S War. 45 War. 96 War. 147 War. 198 War. 249 War. 3OO War. 351 War. 402 mg/kg 1-2O War. 46 War. 97 War. 148 War. 199 War. 250 War. 301 War. 352 War. 403 mg/kg 1-15 War. 47 War. 98 War. 149 War. 200 War. 251 War. 302 War. 353 War. 404 mg/kg 1-10 War. 48 War. 99 War. 150 War. 201 War. 252 War. 303 War. 354 War. 405 mg/kg 1-5 War. 49 War. 100 War. 151 War. 202 War. 253 War. 304 War. 355 War. 406 mg/kg 2-10 War. SO War. 101 War. 152 War. 203 War. 254 War. 305 War. 356 War. 407 mg/kg 2-5 War. 51 War. 102 War. 153 War. 204 War. 255 War. 306 War. 357 War. 408 mg/kg

War. = wariation

TABLE 2 Exemplary combinations of dosage and frequency for methods of treating a CNS disorder by intranasal administration of pooled human IgG. Four Five Six One Two Three Four Five Times Times Times Time Times Times Times Times Weekly Weekly Weekly Daily Daily Daily Daily Daily

O.OS War. 409 War. 460 War. 511 War. 562 War. 613 War. 664 War. 715 War. 766 mg/kg O.1 War. 410 War. 461 War. 512 War. 563 War. 614 War. 665 War. 716 War. 767 mg/kg O.25 War. 411 War. 462 War. 513 War. 564 War. 615 War. 666 War. 717 War. 768 mg/kg O.S War. 412 War. 463 War. 514 War. 565 War. 616 War. 667 War. 718 War. 769 mg/kg 0.75 War. 413 War. 464 War. 515 War. 566 War. 617 War. 668 War. 719 War. 770 mg/kg 1.O War. 414 War. 465 War. 516 War. 567 War. 618 War. 669 War. 72O War. 771 mg/kg 1.5 War. 415 War. 466 War. 517 War. 568 War. 619 War. 670 War. 721 War. 772 mg/kg 2.0 War. 416 War. 467 War. 518 War. 569 War. 620 War. 671 War. 722 War. 773 mg/kg 2.5 War. 417 War. 468 War. 519 War. 570 War. 621 War. 672 War. 723 War. 774 mg/kg US 9,556.260 B2 39 40 TABLE 2-continued Exemplary combinations of dosage and frequency for methods of treating a CNS disorder by intranasal administration of pooled human IgG. Four Five Six One Two Three Four Five Times Times Times Time Times Times Times Times Weekly Weekly Weekly Daily Daily Daily Daily Daily 3.0 War. 418 War. 469 War. 52O War. 571 War. 622 War. 673 War. 724 War. 775 mg/kg 3.5 War. 419 War. 470 War. S21 War. S72 War. 623 War. 674 War. 72S War. 776 mg/kg 4.0 War. 420 War. 471 War. S22 War. S73 War. 624 War. 675 War. 726 War. 777 mg/kg 4.5 War. 421 War. 472 War. S23 War. S74 War. 62S War. 676 War. 727 War. 778 mg/kg S.O War. 422 War. 473 War. S24 War. S75 War. 626 War. 677 War. 728 War. 779 mg/kg 6.O War. 423 War. 474 War. S2S War. S76 War. 627 War. 678 War. 729 War. 780 mg/kg 7.0 War. 424 War. 475 War. S26 War. S77 War. 628 War. 679 War. 730 War. 781 mg/kg 8.0 War. 42S War. 476 War. S27 War. S78 War. 629 War. 680 War. 731 War. 782 mg/kg 9.0 War. 426 War. 477 War. S28 War. S79 War. 630 War. 681 War. 732 War. 783 mg/kg O War. 427 War. 478 War. 529 War. 580 War. 631 War. 682 War. 733 War. 784 mg/kg 1 War. 428 War. 479 War. 530 War. 581 War. 632 War. 683 War. 734 War. 785 mg/kg 2 War. 429 War. 480 War. 531 War. 582 War. 633 War. 684 War. 735 War. 786 mg/kg 3 War. 430 War. 481 War. 532 War. 583 War. 634 War. 685 War. 736 War. 787 mg/kg 4 War. 431 War. 482 War. 533 War. 584 War. 635 War. 686 War. 737 War. 788 mg/kg 5 War. 432 War. 483 War. 534 War. 585 War. 636 War. 687 War. 738 War. 789 mg/kg 6 War. 433 War. 484 War. 535 War. 586 War. 637 War. 688 War. 739 War. 790 mg/kg 7 War. 434 War. 485 War. 536 War. 587 War. 638 War. 689 War. 740 War. 791 mg/kg 8 War. 435 War. 486 War. 537 War. 588 War. 639 War. 690 War. 741 War. 792 mg/kg 9 War. 436 War. 487 War. 538 War. 589 War. 640 War. 691 War. 742 War. 793 mg/kg 2O War. 437 War. 488 War. 539 War. 590 War. 641 War. 692 War. 743 War. 794 mg/kg 22.5 War. 438 War. 489 War. 540 War. 591 War. 642 War. 693 War. 744 War. 795 mg/kg 25 War. 439 War. 490 War. 541 War. 592 War. 643 War. 694 War. 745 War. 796 mg/kg 27.5 War. 440 War. 491 War. 542 War. 593 War. 644 War. 695 War. 746 War. 797 mg/kg 30 War. 441 War. 492 War. 543 War. 594 War. 645 War. 696 War. 747 War. 798 mg/kg 32.5 War. 442 War. 493 War. 544 War. 595 War. 646 War. 697 War. 748 War. 799 mg/kg 35 War. 443 War. 494 War. 545 War. 596 War. 647 War. 698 War. 749 War. 800 mg/kg 37.5 War. 444 War. 495 War. 546 War. 597 War. 648 War. 699 War. 7SO War. 801 mg/kg 40 War. 445 War. 496 War. 547 War. 598 War. 649 War. 7OO War. 751 War. 802 mg/kg 45 War. 446 War. 497 War. 548 War. 599 War. 650 War. 701 War. 752 War. 803 mg/kg 50 War. 447 War. 498 War. 549 War. 600 War. 651 War. 702 War. 753 War. 804 mg/kg OS-40 War. 448 War. 499 War. SSO War. 601 War. 652 War. 703 War. 754 War. 805 mg/kg OS-30 War. 449 War. SOO War. 551 War. 602 War. 653 War. 704 War. 755 War. 806 mg/kg O.S-20 War. 450 War. SO1 War. SS2 War. 603 War. 654 War. 705 War. 756 War. 807 mg/kg O.S-20 War. 451 War. SO2 War. SS3 War. 604 War. 655 War. 706 War. 7S7 War. 808 mg/kg O.S.-10 War. 452 War. SO3 War. SS4 War. 605 War. 656 War. 707 War. 758 War. 809 mg/kg O.S.-S War. 453 War. SO4 War. SSS War. 606 War. 657 War. 708 War. 759 War. 810 mg/kg US 9,556.260 B2 41 42 TABLE 2-continued Exemplary combinations of dosage and frequency for methods of treating a CNS disorder by intranasal administration of pooled human IgG. Four Five Six One Two Three Four Five Times Times Times Time Times Times Times Times Weekly Weekly Weekly Daily Daily Daily Daily Daily 1-2O War. 454 War. SOS War. SS6 War. 607 War. 658 War. 709 War. 760 War. 811 mg/kg 1-15 War. 455 War. SO6 War. SS7 War. 608 War. 659 War. 710 War. 761 War. 812 mg/kg 1-10 War. 456 War. 507 War. 558 War. 609 War. 660 War. 711 War. 762 War. 813 mg/kg 1-5 War. 457 War. 508 War. 559 War. 610 War. 661 War. 712 War. 763 War. 814 mg/kg 2-10 War. 458 War. 509 War. 560 War. 611 War. 662 War. 713 War. 764 War. 815 mg/kg 2-5 War. 459 War. 510 War. 561 War. 612 War. 663 War. 714 War. 765 War. 816 mg/kg

War. = wariation 2O Formulation fluidizers, and tight junction modulators. Specific non-lim Pharmaceutical compositions of pooled human immuno iting examples include bile salts, phospholipids, sodium globulin G described herein can be prepared in accordance glycyrrhetinate, sodium caprate, ammonium tartrate, with methods well known and routinely practiced in the art. gamma. aminolevulinic acid, oxalic acid, malonic acid, See, e.g., Remington: The Science and Practice of Phar 25 Succinic acid, maleic acid, and oxaloacetic acid. macy, Mack Publishing Co., 20" ed., 2000; and Sustained In addition to pooled human IgG, the pharmaceutical and Controlled Release Drug Delivery Systems, J. R. Rob compositions provided herein include one or more stabiliz inson, ed., Marcel Dekker, Inc., New York, 1978. Pharma ing agents. In a specific embodiment, the stabilizing agent is ceutical compositions are preferably manufactured under a buffering agent Suitable for intranasal administration. GMP conditions. Typically, a therapeutically effective dose 30 Non-limiting examples of buffering agents suitable for for or efficacious dose of the pooled human IgG preparation is mulating the pooled human IgG compositions provided employed in the pharmaceutical compositions described herein include an amino acid (e.g., glycine, histidine, or herein. The pharmaceutical composition can be formulated proline) a salt (e.g., citrate, phosphate, acetate, glutamate, into dosage forms by conventional methods known to those 35 tartrate, benzoate, lactate, gluconate, malate. Succinate, for of skill in the art. Dosage regimens are adjusted to provide mate, propionate, or carbonate), or any combination thereof the optimum desired response (e.g., a therapeutic response). adjusted to an appropriate pH. Generally, the buffering agent For example, a single bolus may be administered, several will be sufficient to maintain a suitable pH in the formulation divided doses may be administered over time or the dose for an extended period of time. In a particular embodiment, may be proportionally reduced or increased as indicated by 40 the buffering agent is sufficient to maintain a pH of 4 to 7.5. the exigencies of the therapeutic situation. It can be advan In a specific embodiment, the buffering agent is Sufficient to tageous to formulate parenteral compositions in dosage unit maintain a pH of approximately 4.0, or approximately 4.5, form for ease of administration and uniformity of dosage. or approximately 5.0, or approximately 5.5, or approxi Dosage unit form as used herein refers to physically discrete mately 6.0, or approximately 6.5, or approximately 7.0, or units Suited as unitary dosages for the Subjects to be treated; 45 approximately 7.5. each unit contains a predetermined quantity of active com In a particular embodiment, a pooled human IgG com pound calculated to produce the desired therapeutic effect in position described herein for the treatment of a CNS disor association with the required pharmaceutical carrier. der via intranasal administration contains a stabilizing Actual dosage levels can be varied so as to obtain an amount of an amino acid. In certain embodiments, a stabi amount of the active ingredient which is effective to achieve 50 lizing amount of an amino acid is from about 25 mM to the desired therapeutic response for a particular patient about 500 mM without being toxic to the patient. A physician can start In a particular embodiment, the stabilizing agent doses of the pharmaceutical composition at levels lower than employed in the pooled human IgG compositions provided that required to achieve the desired therapeutic effect and herein is an amino acid. Non-limiting examples of amino gradually increase the dosage until the desired effect is 55 acids include isoleucine, alanine, leucine, asparagine, lysine, achieved. In general, effective doses vary depending upon aspartic acid, methionine, cysteine, phenylalanine, glutamic many different factors, including the specific disease or acid, threonine, glutamine, tryptophan, glycine, Valine, pro condition to be treated, its severity, physiological state of the line, selenocysteine, serine, tyrosine, arginine, histidine, patient, other medications administered, and whether treat ornithine, taurine, combinations thereof, and the like. In one ment is prophylactic or therapeutic. 60 embodiment, the stabilizing amino acids include arginine, In one embodiment, a therapeutic composition of pooled histidine, lysine, serine, proline, glycine, alanine, threonine, human IgG formulated for intranasal administration does not and a combination thereof. In a preferred embodiment, the contain a permeability enhancer. Permeability enhancers amino acid is glycine. In another preferred embodiment, the facilitate the transport of molecules through the mucosa, amino acid is proline. In yet another preferred embodiment, including the mucous, and the nasal epithelium. Non-limit 65 the amino acid is histidine. ing examples of absorption enhancers include mucoadhe For purposes of stabilizing the compositions provided sives, ciliary beat inhibitors, mucous fluidizers, membrane herein, the buffering agent (e.g., glycine, histidine, or pro US 9,556.260 B2 43 44 line) will typically be added to the formulation (or to a mulation is from 4.0 to 7.5. In some embodiments, the pH solution from which a dry powder composition is to be of the histidine formulation is from 4.0 to 6.0. In some prepared) at a concentration from 5 mM to 0.75 M. In one embodiments, the pH of the histidine formulation is from 4.0 embodiment, at least 100 mM of the buffering agent is added to 4.5. In some embodiments, the pH of the histidine to the formulation. In another embodiment, at least 200 mM 5 formulation is from 4.5 to 5.0. In some embodiments, the pH of the buffering agent is added to the formulation. In yet of the histidine formulation is from 4.0 to 5.5. In some another embodiment, at least 250 mM of the buffering agent embodiments, the pH of the histidine formulation is from 4.0 is added to the formulation. In yet other embodiments, the to 6.5. In some embodiments, the pH of the histidine formulations provided herein contains at least 25 mM, 50 formulation is from 4.0 to 7.0. In some embodiments, the pH mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 10 of the histidine formulation is from 4.5 to 6.0. In some mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 embodiments, the pH of the histidine formulation is from 4.5 mM, 650 mM, 700 mM, 750 mM, or more of the buffering to 6.5. In some embodiments, the pH of the histidine agent. In a specific embodiment, the buffering agent is formulation is from 4.5 to 7.0. In some embodiments, the pH glycine. of the histidine formulation is from 4.5 to 7.5. In some In one embodiment, the concentration of buffering agent 15 embodiments, the pH of the histidine formulation is from 5.5 (e.g., glycine, histidine, or proline) in the formulation (or in to 7.0. In some embodiments, the pH of the histidine the solution from which a dry powder composition is to be formulation is from 6.0 to 7.0. In some embodiments, the pH prepared) is at or about from 5 mM to 500 mM. In certain of the histidine formulation is from 6.5 to 7.0. In some embodiments, the concentration of the buffering agent in the embodiments, the pH of the histidine formulation is from 5.0 formulation will be at or about 5 mM, 10 mM, 15 mM, 20 to 6.5. In some embodiments, the pH of the histidine mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, formulation is from 5.0 to 7.0. In some embodiments, the pH 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, of the histidine formulation is from 5.5 to 6.5. In some 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, embodiments, the pH of the histidine formulation is from 6.0 475 mM, 500 mM or higher. In a specific embodiment, the to 6.5. In some embodiments, the pH of the histidine buffering agent is glycine. 25 formulation is from 5.0 to 6.0. In some embodiments, the pH In yet other embodiments, the concentration of the buff of the histidine formulation is from 5.5 to 6.0. In some ering agent (e.g., glycine, histidine, or proline) in formula embodiments, the pH of the histidine formulation is from 5.0 tion (or in the solution from which a dry powder composi to 5.5. In some embodiments, the pH of the histidine tion is to be prepared) is from 50 mM to 500 mM, 100 mM formulation is from 7.0 to 7.5. In some embodiments, the pH to 500 mM, 200 mM to 500 mM, 250 mM to 500 mM, 300 30 of the histidine formulation is from 6.0 to 7.5. In some mM to 500 mM, 50 mM to 300 mM, 100 mM to 300 mM, embodiments, the pH of the histidine formulation is from 5.5 200 mM to 300 mM, or 225 mM to 275 mM. In yet other to 7.5. In some embodiments, the pH of the histidine specific embodiments, the concentration of the buffering formulation is from 5.0 to 7.5. In some embodiments, the pH agent (e.g., glycine, histidine, or proline) in formulations of the histidine formulation is 5.0+0.2, 5.1+0.2, 5.2+0.2, provided herein is 250+50 mM, 250+40 mM, 250+30 mM, 35 5.3+0.2, 5.4+0.2, 5.5+0.2, 5.6+0.2, 5.7+0.2, 5.8+0.2, 250-25 mM, 250-20 mM, 250-15 mM, 250-10 mM, 5.9+0.2, 6.0+0.2, 6.1+0.2, 6.2+0.2, 6.3-0.2, 6.4+0.2, 250-5 mM, or 250 mM. 6.5+0.2, 6.6+0.2, 6.7+0.2, 6.8+0.2, 6.9-0.2, or 7.0+0.2. In In some embodiments, the pooled human immunoglobu some embodiments, the pH of the histidine formulation is lins are formulated with between 100 mM and 400 mM 5.0+0.1, 5.1+0.1, 5.2+0.1, 5.3+0.1, 5.4+0.1, 5.5-0.1, histidine; no more than 10 mM of an alkali metal cation; and 40 5.6+0.1, 5.7+0.1, 5.8+0.1, 5.9+0.1, 6.0+0.1, 6.1+0.1, a pH between 5.0 and 7.0. 6.2+0.1, 6.3-0.1, 6.4+0.1, 6.5-0.1, 6.6+0.1, 6.7+0.1, In some embodiments of the pooled human immuno 6.8+0.1, 6.9+0.1, or 7.0+0.1. globulin histidine formulation, the concentration of histidine In one embodiment, the pooled human IgG compositions is between 5 mM and 500 mM. In another embodiment, the described herein for the treatment of a CNS disorder via concentration of histidine in the formulation will be between 45 intranasal administration is formulated at a pH from about 100 mM and 400 mM. In another embodiment, the concen 4.0 to about 7.0. In particular embodiments, a pooled human tration of histidine in the formulation will be between 200 IgG compositions is formulated at a pH of about 4.0, 4.1. mM and 300 mM. In another embodiment, the concentration 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, of histidine in the formulation will be between 225 mM and 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4., 6.5, 6.7, 6.8, 6.9, or 275 mM. In another embodiment, the concentration of 50 7.0. In other embodiments, a pooled human IgG composi histidine in the formulation will be between 240 mM and tion is formulated at a pH from 4.0 to 6.5, 4.0 to 6.0, 4.0 to 260 mM. In a particular embodiment, the concentration of 5.5, 4.0 to 5.0, 4.0 to 4.5, 4.5 to 6.5, 4.5 to 6.0, 4.5 to 5.5, histidine will be 250 mM. In certain other embodiments, the 4.5 to 5.0. In yet other embodiments, a pooled human IgG concentration of histidine in the formulation will be 5+0.5 composition is formulated at a pH of 4.8+0.5, 4.8+0.4, mM, 10-1 mM, 15-1.5 mM, 20+2 mM, 25+2.5 mM, 50+5 55 4.8+0.3, 4.8+0.2, 4.8+0.1, or about 4.8. mM, 75+7.5 mM, 100-10 mM, 125-12.5 mM, 150-15 mM, In one embodiment, liquid compositions of pooled human 175-17.5 mM, 200+20 mM, 225-22.5 mM, 250-25 mM, IgG formulated for intranasal administration are provided 275-27.5 mM, 300+30 mM, 325-32.5 mM, 350-35 mM, for the treatment of CNS disorders (e.g., Alzheimer's dis 375+37.5 mM, 400-40 mM, 425-42.5 mM, 450-45 mM, ease, Parkinson's disease, and multiple Sclerosis). In a 475+47.5 mM, 500+50 mM or higher. In yet other embodi 60 specific embodiment, the liquid composition is an aqueous ments, the concentration of histidine in the formulation will composition. In a particular embodiment, an aqueous thera be 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM, peutic composition formulated for intranasal administration 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, provided herein consists essentially of a buffering agent and 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, pooled human IgG. 400 mM, 425 mM, 450 mM, 475 mM, 500 mM or higher. 65 In one embodiment, a liquid composition formulated for In some embodiments of the pooled human immuno intranasal administration contains from about 1.0 g pooled globulin histidine formulation, the pH of the histidine for human IgG per liter (g/L IgG) to about 250 g/L IgG. In other US 9,556.260 B2 45 46 embodiments, the liquid composition formulated for intra hyaluronic acid, gelatin, algin, carageenans, carbomers, nasal administration contains about 1 g/L, 2 g/L, 3 g/L, 4 galactomannans, polyethylene glycols, polyvinyl alcohol, g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L. 10 g/L, 12.5g/L, 15 polyvinylpyrrolidone, sodium carboxymethyl dextran, and g/L, 17.5g/L, 20 g/L, 25g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L. Xantham. 50 g/L, 55 g/L, 60 g/L, 65 g/L, 70 g/L, 75 g/L, 80 g/L, 85 In one embodiment, dry powder compositions of pooled g/L, 90 g/L. 95 g/L. 100 g/L, 110 g/L, 120 g/L, 130 g/L, 140 human IgG formulated for intranasal administration are g/L, 150 g/L, 160 g/L, 170 g/L, 180 g/L, 190 g/L, 200 g/L, provided for the treatment of CNS disorders (e.g., Alzheim 210 g/L, 220 g/L, 230 g/L, 240 g/L, 250 g/L, or higher er's disease, Parkinson's disease, and multiple Sclerosis). In concentration of pooled human IgG. In certain embodi a specific embodiment, a dry powder therapeutic composi ments, the liquid composition formulated for intranasal 10 administration contains from 5.0 g/L to 250 g/L. 10 g/L to tion formulated for intranasal administration provided 250 g/L, 20 g/L to 250 g/L, 30 g/L to 250 g/L, 40 g/L to 250 herein consists essentially of a buffering agent and pooled g/L, 50 g/L to 250 g/L, 60 g/L to 250 g/L, 70 g/L to 250 g/L, human IgG. 80 g/L to 250 g/L, 90 g/L to 250 g/L, 100 g/L to 250 g/L, 125 In one embodiment, a dry powder composition of pooled g/L to 250 g/L, 150 g to 250 g/L, 175 g/L to 250 g/L, 200 15 human IgG formulated for intranasal administration further g/L to 250 g/L IgG. comprises a bulking agent. Non-limiting examples of bulk In certain embodiments, the methods for treating a CNS ing agents include oxyethylene maleic anhydride copoly disorder provided herein include intranasally administering mer, polyvinylether, polyvinylpyrrolidone polyvinyl alco a liquid composition containing a low concentration of hol, polyacrylates, including sodium, potassium or pooled human IgG. In one embodiment, a low concentration ammonium polyacrylate, polylactic acid, polyglycolic acid, of pooled human IgG contains from 1.0 g/L to 100 g/L, 5.0 polyvinyl alcohol, polyvinyl acetate, carboxyvinyl polymer, g/L to 100 g/L. 10 g/L to 100 g/L, 20 g/L to 100 g/L, 30 g/L polyvinylpyrrolidone, polyethylene glycol, celluloses (in to 100 g/L, 40 g/L to 100 g/L, 50 g/L to 100 g/L, 60 g/L to cluding cellulose, microcrystalline cellulose, and alpha 100 g/L, 70 g/L to 100 g/L, 75 g/L to 100 g/L, 80 g/L to 100 cellulose), cellulose derivatives (including methyl cellulose, g/L, 1.0 g/L to 50 g/L, 5.0 g/L to 50 g/L. 10 g/L to 50 g/L, 25 ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cel 20 g/L to 50 g/L, 30 g/L to 50 g/L, or 40 g/L to 50 g/L IgG. lulose, hydroxypropyl methyl cellulose, sodium carboxym In certain embodiments, the methods for treating a CNS ethyl cellulose and ethylhydroxy ethyl cellulose), dextrins disorder provided herein include intranasally administering (including alpha-, beta-, or gamma-cyclodeXthn, and dim a liquid composition containing an intermediate concentra ethyl-beta-cyclodexthn), starches (including hydroxyethyl tion of pooled human IgG. In one embodiment, an interme 30 starch, hydroxypropyl Starch, carboxymethyl starch), poly diate concentration of pooled human IgG contains from 75 saccharides (including dextran, dextrin and alginic acid, g/L to 200 g/L, 100 g/L to 200 g/L, 110 g/L to 200 g/L, 120 hyaluronic acid, and pectic acid), carbohydrates (such as g/L to 200 g/L, 130 g/L to 200 g/L, 140 g/L to 200 g/L, 150 mannitol, glucose, lactose, fructose, Sucrose, and amylose), g/L to 200 g/L, 160 g/L to 200 g/L, 170 g/L to 200 g/L, 175 proteins (including casein, gelatin, chitin, and chitosan), g/L to 200 g/L, 180 g/L to 200 g/L, 75 g/L to 150 g/L. 100 35 gums (such as gum arabic, Xanthan gum, tragacanth gum, g/L to 150 g/L, 110 g/L to 150 g/L, 120 g/L to 150 g/L, 130 and glucomannan), phospholipids, and combinations g/L to 150 g/L, or 140 g/L to 150 g/L IgG. thereof. In certain embodiments, the methods for treating a CNS In certain embodiments, a dry powder composition of disorder provided herein include intranasally administering pooled human IgG formulated for intranasal administration a liquid composition containing a high concentration of 40 further comprises a mucosal penetration enhancer. Non pooled human IgG. In one embodiment, a high concentra limiting examples of mucosal penetration enhancers are bile tion of pooled human IgG contains from 175 g/L to 250 g/L, salts, fatty acids, Surfactants and alcohols. Specific non 200 g/L to 250 g/L, 210 g/L to 250 g/L, 220 g/L to 250 g/L, limiting examples of mucosal penetration enhancers are 230 g/L to 250 g/L, or 240 g/L to 250 g/L IgG. Sodium cholate, Sodium dodecyl Sulphate, sodium deoxy In a particular embodiment, a liquid compositions of 45 cholate, taurodeoxycholate, Sodium glycocholate, dimethyl pooled human IgG formulated for intranasal administration sulfoxide or ethanol. consists essentially of from 100 g/L to 250 g/L pooled In certain embodiments, a dry powder composition of human IgG and from 150 mM to 350 mM glycine. pooled human IgG formulated for intranasal administration In another particular embodiment, a liquid compositions further comprises a dispersant. A dispersant is an agent that of pooled human IgG formulated for intranasal administra 50 assists aerosolization of the IgG or the absorption of the IgG tion consists essentially of from 150 g/L to 250 g/L pooled in intranasal mucosal tissue, or both. Non-limiting examples human IgG and from 200 mM to 300 mM glycine. of dispersants are a mucosal penetration enhancers and In yet another particular embodiment, a liquid composi Surfactants. tions of pooled human IgG formulated for intranasal admin In certain embodiments, a dry powder composition of istration consists essentially of from 200 g/L to 250 g/L 55 pooled human IgG formulated for intranasal administration pooled human IgG and 250+25 mM glycine. further comprises a bioadhesive agent. Non-limiting In certain embodiments, the liquid compositions of examples of bioadhesive agents include chitosan or cyclo pooled human IgG formulated for intranasal administration dextrin. In certain embodiments, a dry powder composition provided herein further include a humectant. Non-limiting of pooled human IgG formulated for intranasal administra examples of humectants include glycerin, polysaccharides, 60 tion further comprises a filler. Non-limiting examples of and polyethylene glycols. fillers include Sugars, including lactose, Sucrose, mannitol, In certain embodiments, the liquid compositions of or Sorbitol; cellulose preparations such as: for example, pooled human IgG formulated for intranasal administration maize starch, wheat starch, rice starch, potato starch, gelatin, provided herein further include an agent that increases the gum tragacanth, methylcellulose, microcrystalline cellulose, flow properties of the composition. Non-limiting examples 65 hydroxypropylmethylcellulose, sodium carboxymethylcel of agents that increase to flow properties of an aqueous lulose; or others such as: polyvinylpyrrolidone (PVP or composition include Sodium carboxymethyl cellulose, poVidone) or calcium phosphate. US 9,556.260 B2 47 48 The particle size a dry powder composition of pooled oxymetazoline, tetrahydrozoline, Xylometazoline, clonidine, human IgG can be determined by standard methods in the guanabenz, guanfacine, C.-methyldopa, arginine vasopres art. For example, the particles can be screened or filtered sin, and pseudoephedrine. through a mesh sieve. In certain embodiments, the dry Disorders of the Central Nervous System— particles have an average diameter from about 0.1 um to IVIG treatment has been used in the treatment of CNS about 250 um. In some embodiments, the dry particles have disorders. Specifically, IVIG has been studied or used in the an average diameter between from 1 um to about 25 Lum. In treatment of Multiple Sclerosis (MS), stiff-person syndrome, Some embodiments, the dry particles have an average diam Alzheimer's disease (AD), postpolio syndrome, narcolepsy, eter between from 10 um to about 100 um. In yet other stroke, and fibromyalgia and other pain syndromes. Stangle embodiments, the dray particles have an average diameter of 10 2008 (Therapeutic Advances in Neurological Disorders, 1(2): 115-124). about 0.1 um+10%, 0.2 um+10%, 0.3 um+10%, 0.4 IVIG has also been used to treat neuromyelitis optica um+10%, 0.5 um+10%, 0.6 um+10%, 0.7 um+10%, 0.8 (NMO). NMO, also known as Devic's disease or Devic's um+10%, 0.9 um+10%, 1.0 um+10%, 2 um+10%, 3 syndrome, is an autoimmune, inflammatory disorder of the um+10%, 4 um+10%, 5um+10%, 6 um+10%, 7 um+10%, 15 optic nerves and spinal cord. For example, a 2 g/kg induction 8 um+10%, 9 um+10%, 10 um+10%, 11 um+10%, 12 dose of IVIG followed by 0.4-0.5 g/kg monthly maintenance um+10%, 13 um+10%, 14 um+10%, 15 um+10%, 16 doses of IVIG has been used to treat NMO. Awad et al. 2011 um+10%, 17 um+10%, 18 umi-10%, 19 um+10%, 20 (Current Neuropharmacology, 9:417428). um+10%, 25 um+10%, 30 um+10%, 35 um+10%, 40 IVIG has also been used and studied for the treatment of um+10%, 45 um+10%, 50 um+10%, 60 um+10%. 65 acute disseminated (ADEM). ADEM is um+10%, 70 um+10%, 75 um+10%, 80 um+10%, 85 an immune mediated disease of the brain. Specifically, um+10%, 90 um+10%, 95 um+10%, 100 um+10%, 110 ADEM involves autoimmune demyelination and is classi um+10%, 120 umi-10%, 130 um+10%, 140 um+10%, 150 fied as a MS borderline disease. For example, a standard um+10%, 160 umi-10%, 170 um+10%, 180 um+10%, 190 dose of 2 g/kg IVIG given over 2-5 days can be used to treat um+10%, 200 um+10%, 225 um+10%, 250 um+10%, 275 25 ADEM. Pohl et al. 2012 (Current Treatment Options in um+10%, 300 um+10%, 350 um+10%, 400 um+10%, 450 Neurology, 14:264-275). um+10%, 500 um+10%, or greater. IVIG has also studied and used in the treatment of In one embodiment, gel, cream, or ointment compositions Parkinson's disease (PD). For example, studies have shown of pooled human IgG formulated for intranasal administra that IVIG may reduce C-synuclein neurotoxicity, a possible tion are provided for the treatment of CNS disorders (e.g., 30 contributing factor to the pathogenesis of PD, through an Alzheimer's disease, Parkinson's disease, and multiple scle unknown mechanism. Smith et al. 2012 (International rosis). In a specific embodiment, a gel, cream, or ointment Immunopharmacology, 14:550-557) and Patrias et al. (Clini therapeutic composition formulated for intranasal adminis cal and Experimental Immunology, 161:527-535). tration provided herein consists essentially of a buffering IVIG has also been used and studied for the treatment of agent and pooled human IgG. 35 MS. For example, IVIG has been used successfully in the In one embodiment, a gel, cream, or ointment composi treatment of Schilder's disease (SD), a rare variant of MS. tion of pooled human IgG formulated for intranasal admin Krause et al. 2012 (European J. of Paediatric Neurology, istration further comprises a carrier agent. Non-limiting 16:206-208). IVIG has also been suggested to be beneficial examples of carrier agents for gel and ointment composi in the treatment of acute relapses in MS patients. Elovaara tions include natural or synthetic polymers such as 40 et al. 2011 (Clinical Neuropharmacology, 34(2):84-89). hyaluronic acid, sodium alginate, gelatin, corn starch, gum IVIG has also been used and studied for the treatment of tragacanth, methylcellulose, hydroxyethylcellulose, car obsessive-compulsive disorders (OCD) and tic disorders. boxymethylcellulose, Xanthan gum, dextrin, carboxymeth For example, IVIG was shown to lessen the severity of ylstarch, polyvinyl alcohol, Sodium polyacrylate, methoxy symptoms of OCD and tic disorders in children with infec ethylene maleic anhydride copolymer, polyvinylether, 45 tion-triggered OCD and tic disorders. Perlmutter, et al. 1999 polyvinylpyrrolidone, fats and oils such as beeswax, olive (The Lancet, 354:1153-1158). Similarly, it has been shown oil, cacao butter, Sesame oil, soybean oil, camelia oil, that IVIG is effective in reducing neuropsychiatric symptom peanut oil, beef fat, lard, and lanolin, white petrolatum, severity in a subgroup OCD and tic disorder patients with paraffins, hydrocabon gel ointments, fatty acids such as childhood-onset OCD and tic disorders. Snider et al. 2003 (J. Stearic acid, alcohols such as cetyl alcohol and Stearyl 50 of Child and Adolescent Psychopharmacology, 13(supp 1): alcohol, polyethylene glycol, water, and combinations S81-S88). thereof. In one aspect, the present invention provides a method for In certain embodiments, the pooled human immunoglobu treating a central nervous system (CNS) disorder in a subject lins are co-formulated with one or more vasoconstrictor in need thereof by delivering a therapeutically effective agents. When present, the vasoconstrictor agent reduces 55 amount of a composition comprising pooled human immu non-target exposure (e.g., systemic exposure) of the pooled noglobulin G (IgG) to the brain of the subject, wherein human immunoglobulin, by reducing absorption of the delivering the composition to the brain comprises intrana immunoglobulins into the blood, effectively increasing the Sally administering the composition directly to an epithelium targeting of the immunoglobulin to the CNS (e.g., to the of the nasal cavity of the Subject. In a specific embodiment, brain). Methods for the co-formulation of other pharmaceu 60 the composition is administered directly to the olfactory ticals and vasoconstrictors can be found in U.S. Patent epithelium of the nasal cavity. In certain embodiments, the Application Publication No. 2008/0305077, the content of CNS disorder is selected from the group consisting of a which is expressly incorporated herein by reference in its systemic atrophy primarily affecting the central nervous entirety for all purposes. Non-limiting examples of vaso system, an extrapyramidal and movement disorder, a neu constrictors that may be co-formulated with pooled human 65 rodegenerative disorder of the central nervous system, a immunoglobulins in this fashion include tetrahydrozoline, demyelinating disorder of the central nervous system, an methoxamine, phenylephrine, ephedrine, norepinephrine, episodic or paroxysmal disorder of the central nervous US 9,556.260 B2 49 50 system, a paralytic syndrome of the central nervous system, of spinal cord. In certain embodiments, the CNS disorder is a nerve, nerve root, or plexus disorder of the central nervous disorder characterized by dementia. In certain embodiments, system, an organic mental disorder, a mental or behavioral the dementia is a cortical dementia (associated, for example, disorder caused by psychoactive Substance use, a schizo with Alzheimer's) arising from a disorder affecting the phrenia, Schizotypal, or delusional disorder, a mood (affec- 5 cerebral cortex. In certain embodiments, the dementia is a tive) disorder, neurotic, stress-related, or Somatoform disor Subcortical dementia (associated, for example, with Parkin der, a behavioral syndrome, an adult personality or behavior son's disease and Huntington's disease) resulting from dys disorder, a psychological development disorder, or a child function in the parts of the brain that are beneath the cortex. onset behavioral or emotional disorder. In some embodi In certain embodiments, the dementia is a side effect of drug ments, the CNS disorder is selected from the group consist- 10 administration. In specific embodiments, the dementia is a ing of Alzheimer's disease, Parkinson's disease, multiple side effect of the administration of a chemotherapeutic Sclerosis, amyotrophic lateral Sclerosis (ALS), Huntington's agent. In specific embodiments, the dementia is a result of disease, cerebral palsy, bipolar disorder, Schizophrenia, or undergoing cardiac bypass. In specific embodiments, the Pediatric acute-onset neuropyschiatric syndrome (PANS). In dementia is a result of a vascular disorder (e.g., myocardial some embodiments, the CNS disorder is selected from the 15 , stroke, high blood pressure). In specific embodi group consisting of Alzheimer's disease, Parkinson's dis ments, the dementia is a result of depression. ease, multiple Sclerosis, Pediatric Autoimmune Neuropsy In one embodiment, the CNS disorder is a demyelinating chiatric Disorders Associated with Streptococcal infections disorder of the central nervous system. Non-limiting (PANDAS), or Pediatric acute-onset neuropyschiatric syn examples of demyelinating disorders that affect the central drome (PANS). 2O nervous system include: multiple Sclerosis; an acute dis In one embodiment, the CNS disorder is a systemic seminated demyelination disorder (e.g., neuromyelitis atrophy primarily affecting the central nervous system. Non optica (Devic's syndrome) or acute and Subacute hemor limiting examples of systemic atrophies that primarily affect rhagic leukoencephalitis (Hurst's disease)); diffuse Sclero the central nervous system include: Huntington's disease; sis; central demyelination of corpus callosum, central pon hereditary ataxias (e.g., congenital non-progressive ataxia, 25 tine myelinolysis; acute in demyelinating early-onset cerebellar ataxias—such as early-onset cerebel disease of central nervous system; Subacute necrotizing lar ataxia with essential , Hunt's ataxia, early-onset myelitis; and concentric sclerosis (Baló disease). cerebellar ataxia with retained reflexes, Friedreich's In one embodiment, the CNS disorder is an episodic or ataxia, and X-linked recessive —late paroxysmal disorder of the central nervous system. Non onset cerebellar ataxia, ataxia telangiectasia (Louis-Bar Syn- 30 limiting examples of episodic and paroxysmal disorders that drome), or hereditary spastic ); a spinal muscular affect the central nervous system include: (e.g., atrophy or related disorder thereof (e.g., Werdnig-Hoffman localization-related (focal) (partial) idiopathic epilepsy and disease (Type 1), of childhood epileptic syndromes with of localized onset, local (Fazio-Londe syndrome), Kugelberg-Welander disease ization-related (focal) (partial) symptomatic epilepsy and (Type 3), or a —such as familial motor 35 epileptic syndromes with simple partial seizures; localiza neuron disease, amyotrophic lateral Sclerosis (ALS), pri tion-related (focal)(partial) symptomatic epilepsy and epi mary lateral Sclerosis, progressive bulbar palsy, and progres leptic syndromes with complex partial seizures; a benign sive spinal muscular atrophy); paraneoplastic neuromyopa epileptic syndrome—such as in infancy thy and neuropathy; Systemic atrophy primarily affecting the and neonatal convulsions (familial)—childhood absence central nervous system in neoplastic disease; paraneoplastic 40 epilepsy (e.g., pyknolepsy), epilepsy with grand mal sei limbic ; and systemic atrophy primarily Zures on awakening, a juvenile epilepsy—such as absence affecting the central nervous system in myxoedema. epilepsy or myoclonic epilepsy (impulsive petit mal)—a In one embodiment, the CNS disorder is an extrapyrami nonspecific epileptic seizure—such as an atonic, clonic, dal and movement disorder. Non-limiting examples of myoclonic, tonic, or tonic-clonic epileptic seizure, epilepsy extrapyramidal and movement disorders that affect the cen- 45 with myoclonic absences or myoclonic-astatic seizures, tral nervous system include: Parkinson's disease; a second infantile spasms, Lennox-Gastaut syndrome, Salaam ary (e.g., malignant neuroleptic syndrome or attacks, symptomatic early myoclonic encephalopathy, postencephalitic parkinsonism); a degenerative disease of West's syndrome, epilepsia partialis continua (Kozhevnikov the basal ganglia (e.g., Hallervorden-Spatz disease, progres epilepsy), grand mal seizures, or petit mal); (e.g., sive Supranuclear ophthalmoplegia (Steele-Richardson- 50 a migraine—Such as a migraine without aura (common Olszewski disease), or striatonigral degeneration), a dysto migraine), a migraine with aura (classical migraine), status nia (e.g., drug-induced , idiopathic familial migrainosus, and complicated migraine— dystonia, idiopathic non-familial dystonia, spasmodic torti syndrome, a vascular headache, a tension-type headache, a collis, idiopathic orofacial dystonia—such as orofacial dys chronic post-traumatic headache, or a drug-induced head kinesia—or ); an , a drug- 55 ache); a cerebrovascular episodic or paroxysmal disorder induced tremor, , drug-induced , drug (e.g., a transient cerebral ischaemic attacks or related Syn induced tics; ; and stiff-man drome—such as vertebrobasilar syndrome, carotid syndrome. artery syndrome (hemispheric), a multiple and bilateral In one embodiment, the CNS disorder is a neurodegen precerebral artery syndrome, amaurosis fugax, and transient erative disorder of the central nervous system. Non-limiting 60 global amnesia—a vascular syndrome of the brain—such as examples of neurodegenerative disorders that affect the syndrome, anterior cerebral artery central nervous system include: Alzheimer's disease; a cir syndrome, posterior cerebral artery syndrome, a brain stem cumscribed brain atrophy (e.g., Pick's disease); senile stroke syndrome (e.g., Benedikt Syndrome, Claude Syn degeneration of brain; a degeneration of nervous system due drome, Foville syndrome, Millard-Gubler syndrome, Wal to alcohol; grey-matter degeneration (e.g., Alpers disease); 65 lenberg syndrome, or Weber syndrome), cerebellar stroke Lewy body dementia, Subacute necrotizing encephalopathy syndrome, pure motor lacunar syndrome, pure sensory lacu (e.g., Leigh's disease); and Subacute combined degeneration nar syndrome, or a lacunar syndromes); and a US 9,556.260 B2 51 52 (e.g., insomnia, hyperinsomnia, a disruption in circadian and dysfunction (e.g., organic personality disorder, posten rhythm, sleep apnoea, narcolepsy, or cataplexy). cephalitic syndrome, or postconcussional syndrome). In one embodiment, the CNS disorder is a paralytic In one embodiment, the CNS disorder is a mental or syndrome of the central nervous system. Non-limiting behavioral disorder caused by psychoactive Substance use. examples of paralytic syndromes that affect the central Non-limiting examples of mental or behavioral disorders nervous system include: a cerebral palsy (e.g., spastic quad caused by psychoactive Substance use that affect the central riplegic cerebral palsy, spastic diplegic cerebral palsy, spas nervous system include: acute intoxication (e.g., from alco tic hemiplegic cerebral palsy, dyskinetic cerebral palsy, or hol, opioid, cannabis, benzodiazepine, or cocaine use); a ataxic cerebral palsy); a hemiplegia (e.g., flaccid hemiplegia dependence syndrome (e.g., from alcohol, opioid, cannabis, or spastic hemiplegia); a paraplegia or (e.g., 10 benzodiazepine, cocaine, or nicotine addiction); a with flaccid paraplegia, spastic paraplegia, paralysis of both drawal syndrome (e.g., an alcohol or benzodiazepine with lower limbs, lower paraplegia, flaccid tetraplegia, spastic drawal syndrome); delirium tremens; and a psychotic dis tetraplegia, or quadriplegia); diplegia of upper limbs; order (e.g., alcoholic hallucinosis or stimulant psychosis); an monoplegia of a lower limb, monoplegia of an upper limb; amnesic syndrome (e.g., Korsakoffs syndrome); a residual ; and Todd's paralysis (postepilep 15 and late-onset psychotic disorder (e.g., posthallucinogen tic). perception disorder). In one embodiment, the CNS disorder is a nerve, nerve In one embodiment, the CNS disorder is an autism root, or plexus disorder of the central nervous system. spectrum disorder. In certain embodiments, the CNS disor Non-limiting examples of nerve, nerve root, or plexus der is autism, Asperger syndrome, pervasive developmental disorders that affect the central nervous system include: a disorder not otherwise specified (PDD-NOS), childhood disorder of the trigeminal nerve (V; e.g., trigeminal neural disintegrative disorder, or Rett syndrome. gia); a facial nerve disorders (VII; e.g., bell's palsy, facial In one embodiment, the CNS disorder is a schizophrenia, palsy, geniculate ganglionitis, melkersson’s syndrome, Schizotypal, or delusional disorder. Non-limiting examples melkersson-Rosenthal syndrome, a clonic hemifacial spasm, of Schizophrenia, Schizotypal, and delusional disorders that facial myokymia); a disorder of the olfactory nerve (I); a 25 affect the central nervous system include: Schizophrenia disorder of the glossopharyngeal nerve (IX); a disorder of (e.g., paranoid Schizophrenia, hebephrenic schizophrenia the vagus nerve (X); a disorder of the hypoglossal nerve (disorganized schizophrenia), catatonic schizophrenia, (XII); a disorder of multiple cranial nerves; and a nerve root undifferentiated Schizophrenia, post-Schizophrenic depres or plexus disorder affecting the CNS (e.g., a brachial plexus Sion, residual schizophrenia, simple schizophrenia, cenest disorder—such as thoracic outlet syndrome—a lumbosacral 30 hopathic schizophrenia, Schizophreniform disorder, or plexus disorder, a cervical root, a thoracic root disorder, a Schizophreniform psychosis); Schizotypal disorder, a persis lumbosacral root disorder, a neuralgic amyotrophy—such as tent delusional disorder (e.g., delusional disorder, delusional Parsonage-Aldren-Turner syndrome—orphantom limb Syn dysmorphophobia, involutional paranoid state, or paranoia drome with or without pain). querulans); an acute or transient psychotic disorder (e.g., In one embodiment, the CNS disorder is an otherwise 35 acute polymorphic psychotic disorder without symptoms of classified disorder of the central nervous system. Non Schizophrenia, acute polymorphic psychotic disorder with limiting examples of these disorders include: hydrocepha symptoms of Schizophrenia, or acute schizophrenia-like lus; a , a cerebral cyst; anoxic brain psychotic disorder); an induced delusional disorder (e.g., damage; benign intracranial hypertension; postviral fatigue folie a deux, induced paranoid disorder, or induced psy syndrome; an encephalopathy; compression of brain; cere 40 chotic disorder); a schizoaffective disorder (e.g., manic type, bral oedema; reye's syndrome; postradiation encephalopa depressive type, or mixed type schizoaffective disorder); and thy; traumatic brain injury; : ; a chronic hallucinatory psychosis. vascular myelopathy; ; myelopathy; In one embodiment, the CNS disorder is a mood (affec a leak; a disorder of the meninges (e.g., tive) disorder. Non-limiting examples of mood (affective) cerebral or spinal meningeal adhesion); and a post-proce 45 disorders that affect the central nervous system include: a dural disorder of nervous system (e.g., cerebrospinal fluid manic episode (e.g., hypomania, mania without psychotic leak from spinal puncture, an adverse reaction to a spinal or symptoms, or mania with psychotic symptoms); a bipolar lumbar puncture, or intracranial hypotension following ven affective disorder (e.g., bipolar affective disorder—current tricular shunting). episode hypomanic, bipolar affective disorder—current epi In one embodiment, the CNS disorder is an organic 50 sode manic without psychotic symptoms, bipolar affective mental disorder. Non-limiting examples of organic mental disorder—current episode manic with psychotic symptoms, disorders that affect the central nervous system include: bipolar affective disorder—current episode mild or moder dementia (e.g., dementia associated with Alzheimer's dis ate depression, bipolar affective disorder—current episode ease, Pick's disease, Creutzfeldt-Jakob disease, Hunting severe depression without psychotic symptoms, bipolar ton's disease, Parkinson's disease, or human immunodefi 55 affective disorder—current episode severe depression with ciency virus (HIV) disease, or —such as psychotic symptoms, bipolar affective disorder—current multi-infarct dementia); organic amnesic syndrome not episode mixed, bipolar affective disorder—currently in induced by alcohol and other psychoactive Substances); remission, bipolar II disorder, or recurrent manic episodes); delirium not induced by alcohol and other psychoactive a depressive episode (e.g., mild depressive episode, moder Substances; a mental disorder due to brain damage and 60 ate depressive episode, severe depressive episode without dysfunction and to physical disease (e.g., organic halluci psychotic symptoms, severe depressive episode with psy nosis, organic catatonic disorder, organic delusional (schizo chotic symptoms, atypical depression, or single episodes of phrenia-like) disorder, organic mood (affective) disorder, “masked depression); a recurrent depressive disorder (e.g., organic anxiety disorder, organic dissociative disorder; recurrent depressive disorder—current episode mild, recur organic emotionally labile (asthenic) disorder; a mild cog 65 rent depressive disorder—current episode moderate, recur nitive disorder, or organic brain syndrome); and a person rent depressive disorder—current episode severe without ality and behavioral disorders due to brain disease, damage psychotic symptoms, recurrent depressive disorder—current US 9,556.260 B2 53 54 episode severe with psychotic symptoms, or recurrent bling, pathological fire-setting (pyromania), pathological depressive disorder—currently in remission); a persistent stealing (kleptomania), or trichotillomania); and mood (affective) disorder (e.g., cyclothymia or dysthymia); Munchausen syndrome. mixed affective episode; and recurrent brief depressive In one embodiment, the CNS disorder is a psychological episodes. development disorder. Non-limiting examples of psycho In one embodiment, the CNS disorder is a neurotic, logical development disorders that affect the central nervous stress-related, or somatoform disorder. Non-limiting system include: a developmental disorder of speech or examples of neurotic, stress-related, or somatoform disor language (e.g., specific speech articulation disorder, expres ders that affect the central nervous system include: a phobic sive language disorder, receptive language disorder (recep 10 tive aphasia), acquired aphasia with epilepsy (Landau-Kl anxiety disorder (e.g., agoraphobia, anthropophobia, social effner disorder), or lisping); a developmental disorder of neurosis, acrophobia, animal phobias, claustrophobia, or scholastic skills (e.g., a specific reading disorder—such as simple phobia); an otherwise categorized anxiety disorder developmental dyslexia—specific spelling disorder, a spe (e.g., panic disorder (episodic paroxysmal anxiety) or gen cific disorder of arithmetical skills—such as developmental eralized anxiety disorder); obsessive-compulsive disorder, 15 acalculia and Gerstmann syndrome—or a mixed disorder of an adjustment disorder (e.g., acute stress reaction; post scholastic skills); a developmental disorder of motor func traumatic stress disorder, or adjustment disorder); a disso tion (e.g., developmental dyspraxia); a mixed specific devel ciative (conversion) disorder (e.g., dissociative amnesia, opmental disorder, and a pervasive developmental disorder dissociative fugue, dissociative stupor; trance disorder, pos (e.g., childhood autism, atypical autism, Rett's syndrome, session disorder, dissociative motor disorder, dissociative overactive disorder associated with mental retardation and convulsions, dissociative anaesthesia and sensory loss, Stereotyped movements, or Asperger's syndrome). mixed dissociative (conversion) disorder, Ganser's Syn In one embodiment, the CNS disorder is a behavioral or drome, or multiple personality disorder); a Somatoform emotional disorder with onset usually occurring in child disorder (e.g., Briquet's disorder, multiple psychosomatic hood and adolescence. Non-limiting examples of behavioral disorder, a hypochondriacal disorder—such as body dys 25 or emotional disorders with onset usually occurring in morphic disorder, dysmorphophobia (nondelusional), hypo childhood and adolescence that affect the central nervous chondriacal neurosis, hypochondriasis, and nosophobia—a system include: a hyperkinetic disorder (e.g., a disturbance Somatoform autonomic dysfunction—such as cardiac neu of activity and attention—such as attention-deficit hyperac rosis, Da Costa's syndrome, gastric neurosis, and neurocir tivity disorder and attention deficit syndrome with hyperac culatory asthenia—or psychalgia); neurasthenia; deperson 30 tivity—or hyperkinetic conduct disorder); a conduct disor alization-derealization syndrome; Dhat syndrome, der (e.g., conduct disorder confined to the family context, occupational neurosis (e.g., writer's cramp); psychasthenia: unsocialized conduct disorder, socialized conduct disorder, psychasthenic neurosis; and psychogenic syncope. or oppositional defiant disorder); a mixed disorder of con In one embodiment, the CNS disorder is a behavioral duct or emotions (e.g., depressive conduct disorder); an syndrome associated with physiological disturbances or 35 emotional disorder with onset specific to childhood (e.g., physical factors. Non-limiting examples of behavioral Syn separation anxiety disorder of childhood, phobic anxiety dromes associated with physiological disturbances or physi disorder of childhood, social anxiety disorder of childhood, cal factors that affect the central nervous system include: an sibling rivalry disorder, identity disorder, or overanxious eating disorder (e.g., anorexia nervos, atypical anorexia disorder); a disorder of social functioning with onset specific nervosa, bulimia nervosa, atypical bulimia nervosa, over 40 to childhood and adolescence (e.g., elective mutism, reactive eating associated with other psychological disturbances, attachment disorder of childhood, or disinhibited attachment Vomiting associated with other psychological disturbances, disorder of childhood); a tic disorder (e.g., transient tic or pica in adults); a nonorganic sleep disorder (e.g., nonor disorder, chronic motor or vocal tic disorder, or combined ganic insomnia, nonorganic hypersomnia, nonorganic dis vocal and multiple motor tic disorder (de la Tourette); and an order of the sleep-wake schedule, sleepwalking (Somnam 45 otherwise classified behavioral or emotional disorder with bulism), sleep terrors (night terrors), or nightmares); a onset usually occurring in childhood and adolescence (e.g., sexual dysfunction not caused by organic disorder or dis nonorganic enuresis, nonorganic encopresis, feeding disor ease; a mental or behavioral disorder associated with the der of infancy and childhood, pica of infancy and childhood, puerperium (e.g., postnatal depression, postpartum depres Stereotyped movement disorders, Stuttering (stammering), Sion, or puerperal psychosis); and abuse of non-dependence 50 cluttering, attention deficit disorder without hyperactivity, producing Substances. Pediatric Autoimmune Neuropsychiatric Disorders Associ In one embodiment, the CNS disorder is an adult person ated with Streptococcal infections (PANDAS), or Pediatric ality or behavior disorder. Non-limiting examples of adult acute-onset neuropyschiatric syndrome (PANS)). personality and behavior disorders that affect the central In one embodiment of the method for treating a CNS nervous system include: a specific personality disorder (e.g., 55 disorder, the method includes intranasally administering a paranoid personality disorder, Schizoid personality disorder, dry powder composition containing from 0.05 mg/kg to 50 a dissocial personality disorder—such as antisocial person mg/kg pooled human immunoglobulin to a Subject in need ality disorder—an emotionally unstable personality disor thereof daily. In other embodiments, the methods provided der—such as borderline personality disorder histrionic herein for the treatment of a CNS disorder include intrana personality disorder, an anankastic personality disorder— 60 Sally administering a dry powder composition of pooled Such as obsessive-compulsive personality disorder, anxious human IgG in a dosage/frequency combination selected (avoidant) personality disorder, dependent personality dis from variations 1 to 816 found in Table 1 and Table 2. In a order, eccentric personality disorder, haltlose personality particular embodiment, the method comprises administering disorder, immature personality disorder, narcissistic person the dry powder composition directly to a nasal epithelium of ality disorder, passive-aggressive personality disorder, or 65 the Subject. In a particular embodiment, the method com psychoneurotic personality disorder); mixed personality dis prises administering the dry powder composition directly to order, a habit or impulse disorder (e.g., pathological gam the olfactory epithelium of the subject. US 9,556.260 B2 55 56 In one embodiment of the method for treating a CNS thereof daily. In other embodiments, the methods provided disorder, the method includes intranasally administering a herein for the treatment of Alzheimer's disease include liquid (e.g., an aqueous) composition containing from 0.05 intranasally administering a dry powder composition of mg/kg to 50 mg/kg pooled human immunoglobulin to a pooled human IgG in a dosage/frequency combination subject in need thereof daily. In other embodiments, the selected from variations 1 to 816 found in Table 1 and Table methods provided herein for the treatment of a CNS disorder 2. In a particular embodiment, the method comprises admin include intranasally administering a liquid (e.g., an aqueous) istering the dry powder composition directly to a nasal composition of pooled human IgG in a dosage/frequency epithelium of the subject. In a particular embodiment, the combination selected from variations 1 to 816 found in Table method comprises administering the dry powder composi 1 and Table 2. In a particular embodiment, the method 10 comprises administering the composition drop-wise directly tion directly to the olfactory epithelium of the subject. In one to a nasal epithelium of the Subject. In a particular embodi embodiment, the Alzheimer's disease is early-onset ment, the method comprises administering the composition Alzheimer's disease. In another embodiment, the Alzheim drop-wise directly to the olfactory epithelium of the subject. er's disease is late-onset Alzheimer's disease. In another particular embodiment, the method comprises 15 In one embodiment of the method for treating Alzheim administering the composition via a spray directly to a nasal er's disease, the method includes intranasally administering epithelium of the subject. In a particular embodiment, the a liquid (e.g., an aqueous) composition containing from 0.05 method comprises administering the composition via a spray mg/kg to 50 mg/kg pooled human immunoglobulin to a directly to the olfactory epithelium of the subject. subject in need thereof daily. In other embodiments, the In one embodiment of the method for treating a CNS methods provided herein for the treatment of Alzheimer's disorder, the method includes intranasally administering a disease include intranasally administering a liquid (e.g., an gel, cream, or ointment composition containing from 0.05 aqueous) composition of pooled human IgG in a dosage/ mg/kg to 50 mg/kg pooled human immunoglobulin to a frequency combination selected from variations 1 to 816 subject in need thereof daily. In other embodiments, the found in Table 1 and Table 2. In a particular embodiment, the methods provided herein for the treatment of a CNS disorder 25 method comprises administering the composition drop-wise include intranasally administering a gel, cream, or ointment directly to a nasal epithelium of the Subject. In a particular composition of pooled human IgG in a dosage/frequency embodiment, the method comprises administering the com combination selected from variations 1 to 816 found in Table position drop-wise directly to the olfactory epithelium of the 1 and Table 2. In a particular embodiment, the method Subject. In another particular embodiment, the method com comprises administering the gel, cream, or ointment com 30 prises administering the composition via a spray directly to position directly to a nasal epithelium of the Subject. In a a nasal epithelium of the Subject. In a particular embodi particular embodiment, the method comprises administering ment, the method comprises administering the composition the gel, cream, or ointment composition directly to the via a spray directly to the olfactory epithelium of the subject. olfactory epithelium of the subject. In one embodiment, the Alzheimer's disease is early-onset Alzheimer's Disease 35 Alzheimer's disease. In another embodiment, the Alzheim IVIG has been used in the treatment of Alzheimer's er's disease is late-onset Alzheimer's disease. disease. It has been proposed that WIG contains antibodies In one embodiment of the method for treating Alzheim against B-amyloid. Relkin et al. 2009 (Neurobiol. Aging er's disease, the method includes intranasally administering 30(11): 1728-36). In this study, pooled human IgG was a gel, cream, or ointment composition containing from 0.05 administered intravenously (IVIG therapy) to eight subjects 40 mg/kg to 50 mg/kg pooled human immunoglobulin to a diagnosed with mild Alzheimer's disease (AD). The patients subject in need thereof daily. In other embodiments, the received IVIG therapy for 6 months, discontinued treatment, methods provided herein for the treatment of Alzheimer's and then resumed treatment for 9 more months. It was found disease include intranasally administering a gel, cream, or that B-amyloid antibodies in the serum from AD patients ointment composition of pooled human IgG in a dosage/ increased in proportion to IVIG dose and plasma levels of 45 frequency combination selected from variations 1 to 816 B-amyloid increased transiently after each infusion. After 6 found in Table 1 and Table 2. In a particular embodiment, the months of treatment, mini-mental state tests were performed method comprises administering the gel, cream, or ointment on the patients. The mini-mental state scores increased an composition directly to a nasal epithelium of the Subject. In average of 2.5 points after 6 months, returned to baseline a particular embodiment, the method comprises administer during washout and remained stable during Subsequent IVIG 50 ing the gel, cream, or ointment composition directly to the treatment. olfactory epithelium of the subject. In one embodiment, the In one aspect, the present invention provides a method for Alzheimer's disease is early-onset Alzheimer's disease. In treating Alzheimer's disease in a subject in need thereof by another embodiment, the Alzheimer's disease is late-onset delivering a therapeutically effective amount of a composi Alzheimer's disease. tion comprising pooled human immunoglobulin G (IgG) to 55 Multiple Sclerosis the brain of the subject, wherein delivering the composition Multiple sclerosis (MS) is a chronic neurodegenerative to the brain comprises intranasally administering the com and inflammatory disease of the central nervous system position directly to an epithelium of the nasal cavity of the (CNS) that represents one of the most prevalent human Subject. In a specific embodiment, the composition is admin autoimmune diseases. Multiple sclerosis (MS) is an auto istered directly to the olfactory epithelium of the nasal 60 immune disease that specifically affects the brain and spinal cavity. In one embodiment, the Alzheimer's disease is cord. MS is caused by damage to the myelin sheath, the early-onset Alzheimer's disease. In another embodiment, the protective covering that surrounds nerve cells. When the Alzheimer's disease is late-onset Alzheimer's disease. myelin sheath is damaged, nerve signals slow down or stop. In one embodiment of the method for treating Alzheim Damage to the myelin sheath is caused by inflammation er's disease, the method includes intranasally administering 65 which occurs when the body's own immune cells attack the a dry powder composition containing from 0.05 mg/kg to 50 nervous system. This can occur along any area of the brain, mg/kg pooled human immunoglobulin to a subject in need optic nerve, and spinal cord. US 9,556.260 B2 57 58 MS is classified into four subtypes based on the diseases from variations 1 to 816 found in Table 1 and Table 2. In a progression: Relapsing-Remitting MS (RMSS), Secondary particular embodiment, the method comprises administering Progressive MS (SPMS), Primary-Progressive MS (PPMS), the dry powder composition directly to a nasal epithelium of and Progressive-Relapsing MS (PRMS). More than 80 per the Subject. In a particular embodiment, the method com cent of patients who are diagnosed with MS exhibit initial prises administering the dry powder composition directly to signs of RMSS. RMSS is characterized by relapse (charac the olfactory epithelium of the subject. terized by symptom flare-ups) followed by remission. The In one embodiment of the method for treating multiple relapses can be mild to severe flare-ups and the remissions Sclerosis, the method includes intranasally administering a can last for days to months. RMSS patients often develop liquid (e.g., an aqueous) composition containing from 0.05 SPMS. SPMS is characterized by relapses followed by only 10 mg/kg to 50 mg/kg pooled human immunoglobulin to a partial recoveries. During the partial recovery phase, the subject in need thereof daily. In other embodiments, the symptoms may lessen but do not go into full remission. methods provided herein for the treatment of multiple scle SPMS is a progressive subtype of MS wherein the symp rosis include intranasally administering a liquid (e.g., an toms steadily worsen until a chronic disability replaces the aqueous) composition of pooled human IgG in a dosage/ cycles of recovery and partial recovery. PPMS accounts for 15 frequency combination selected from variations 1 to 816 approximately 15 percent of MS occurrences. It is charac found in Table 1 and Table 2. In a particular embodiment, the terized by a slow and steady progression without periods of method comprises administering the composition drop-wise remission or partial recovery. PRMS is the least common directly to a nasal epithelium of the Subject. In a particular subtype of MS. PRMS is characterized by steadily worsen embodiment, the method comprises administering the com ing symptoms and attacks followed by periods of remission. position drop-wise directly to the olfactory epithelium of the There are peptide-induced and transgenic mouse model Subject. In another particular embodiment, the method com for MS. Experimental autoimmune encephalomyelitis prises administering the composition via a spray directly to (EAE) is an animal model of brain inflammation. EAE is an a nasal epithelium of the Subject. In a particular embodi inflammatory of the CNS. Acute and ment, the method comprises administering the composition relapsing EAE is characterized by the formation of focal 25 via a spray directly to the olfactory epithelium of the subject. inflammatory demyelinating lesions in the of In one embodiment of the method for treating multiple the brain. This phenotype can be induced in normal SJL Sclerosis, the method includes intranasally administering a mice through the administration of PLP139-151 peptide. gel, cream, or ointment composition containing from 0.05 Chronic progressive EAE is pathologically associated with mg/kg to 50 mg/kg pooled human immunoglobulin to a a widespread axonal damage in the normal appearing white 30 subject in need thereof daily. In other embodiments, the matter and massive demyelination in the grey matter, par methods provided herein for the treatment of multiple scle ticularly in the cortex. This phenotype can be induced in rosis include intranasally administering a gel, cream, or normal C57BL/6 mice through the administration of ointment composition of pooled human IgG in a dosage/ MOG35-55 peptide. frequency combination selected from variations 1 to 816 There is also evidence that tumor factor (TNF) 35 found in Table 1 and Table 2. In a particular embodiment, the ligand/receptor superfamily, particularly TNF and Fas/Fas method comprises administering the gel, cream, or ointment ligand (FasL) are involved in the pathogenesis of MS. composition directly to a nasal epithelium of the Subject. In Akassoglou et al. 1998 (Am J Pathol. 153(3): 801-813). a particular embodiment, the method comprises administer Accordingly, mouse models deficient in TNF can be used to ing the gel, cream, or ointment composition directly to the study the pathologies of MS. The genotype of transgenic 40 olfactory epithelium of the subject. TNF knockout mouse models include p55TNFR (p55-/-), Parkinson's Disease p75TNFR (p75-/-), and TNF (TNF-/-). Parkinson's disease (PD) is a degenerative disorder of the IVIG has proven useful in the treatment of a number of CNS. PD is notably linked to a decrease in motor control. autoimmune diseases; however its role in the treatment of The loss of motor control caused by PD results from the MS remains uncertain. IVIG trials in different types of MS 45 death of dopamine-generating cells in the Substantia nigra, a patients have produced variable results ranging from reports region of the midbrain. Early in the progression of the of monthly IVIG being beneficial to IVIG administration not disease, the most common symptoms include shaking, rigid slowing disease progression or reversing disease-induced ity, slowness of movement and difficulty with walking and deficits. gait. As the disease progresses, cognitive and behavioral In one aspect, the present invention provides a method for 50 problems arise, with dementia occurring in the advanced treating multiple Sclerosis in a subject in need thereof by stages of the disease. Additional symptoms include sensory, delivering a therapeutically effective amount of a composi sleep and emotional problems. PD is more common in the tion comprising pooled human immunoglobulin G (IgG) to elderly, with symptoms most commonly occurring after the the brain of the subject, wherein delivering the composition age of 50. to the brain comprises intranasally administering the com 55 There are numerous transgenic mouse models for PD. position directly to an epithelium of the nasal cavity of the These models include, for example, Park2 (parkin) trans Subject. In a specific embodiment, the composition is admin genic strains, LRRK2 transgenic strains, and Synuclein istered directly to the olfactory epithelium of the nasal transgenic strains (Jackson Laboratories, Bar Harbor, Me.). cavity. In addition to transgenic models, parkinsonian symptoms In one embodiment of the method for treating multiple 60 can also be induced in mice by administering the compounds Sclerosis, the method includes intranasally administering a MPTP, rotenone, paraquat, or maneb. dry powder composition containing from 0.05 mg/kg to 50 In one aspect, the present invention provides a method for mg/kg pooled human immunoglobulin to a subject in need treating Parkinson's disease in a subject in need thereof by thereof daily. In other embodiments, the methods provided delivering a therapeutically effective amount of a composi herein for the treatment of multiple sclerosis include intra 65 tion comprising pooled human immunoglobulin G (IgG) to nasally administering a dry powder composition of pooled the brain of the subject, wherein delivering the composition human IgG in a dosage/frequency combination selected to the brain comprises intranasally administering the com US 9,556.260 B2 59 60 position directly to an epithelium of the nasal cavity of the In one embodiment of the first aspect, at least 60% of the Subject. In a specific embodiment, the composition is admin pooled human IgG administered to the Subject contacts the istered directly to the olfactory epithelium of the nasal nasal epithelium of the subject. cavity. In one embodiment of the first aspect, the nasal epithelium In one embodiment of the method for treating Parkinson's is the olfactory epithelium of the subject. disease, the method includes intranasally administering a In one embodiment of the first aspect, at least 40% of the dry powder composition containing from 0.05 mg/kg to 50 pooled human IgG administered to the Subject contacts the mg/kg pooled human immunoglobulin to a subject in need olfactory epithelium of the subject. thereof daily. In other embodiments, the methods provided In one embodiment of the first aspect, at least 50% of the herein for the treatment of Parkinson's disease include 10 pooled human IgG administered to the Subject contacts the intranasally administering a dry powder composition of olfactory epithelium of the subject. pooled human IgG in a dosage/frequency combination In one embodiment of the first aspect, at least 60% of the selected from variations 1 to 816 found in Table 1 and Table pooled human IgG administered to the Subject contacts the 2. In a particular embodiment, the method comprises admin olfactory epithelium of the subject. istering the dry powder composition directly to a nasal 15 In one embodiment of the first aspect, the nasal epithelium epithelium of the subject. In a particular embodiment, the is a nasal epithelium of the Subject associated with trigemi method comprises administering the dry powder composi nal nerve endings. tion directly to the olfactory epithelium of the subject. In one embodiment of the first aspect, at least 40% of the In one embodiment of the method for treating Parkinson's pooled human IgG administered to the Subject contacts the disease, the method includes intranasally administering a nasal epithelium of the Subject associated with trigeminal liquid (e.g., an aqueous) composition containing from 0.05 nerve endings. mg/kg to 50 mg/kg pooled human immunoglobulin to a In one embodiment of the first aspect, at least 50% of the subject in need thereof daily. In other embodiments, the pooled human IgG administered to the Subject contacts the methods provided herein for the treatment of Parkinson's nasal epithelium of the Subject associated with trigeminal disease include intranasally administering a liquid (e.g., an 25 nerve endings. aqueous) composition of pooled human IgG in a dosage/ In one embodiment of the first aspect, at least 60% of the frequency combination selected from variations 1 to 816 pooled human IgG administered to the Subject contacts the found in Table 1 and Table 2. In a particular embodiment, the nasal epithelium of the Subject associated with trigeminal method comprises administering the composition drop-wise nerve endings. directly to a nasal epithelium of the Subject. In a particular 30 In one embodiment of the first aspect, delivering the embodiment, the method comprises administering the com composition to the brain comprises intranasally administer position drop-wise directly to the olfactory epithelium of the ing the composition to the upper third of the nasal cavity of Subject. In another particular embodiment, the method com the subject. prises administering the composition via a spray directly to In one embodiment of the first aspect, at least 40% of the a nasal epithelium of the Subject. In a particular embodi 35 pooled human IgG administered to the Subject contacts the ment, the method comprises administering the composition upper third of the nasal cavity of the subject. via a spray directly to the olfactory epithelium of the subject. In one embodiment of the first aspect, at least 50% of the In one embodiment of the method for treating Parkinson's pooled human IgG administered to the Subject contacts the disease, the method includes intranasally administering a upper third of the nasal cavity of the subject. gel, cream, or ointment composition containing from 0.05 40 In one embodiment of the first aspect, at least 60% of the mg/kg to 50 mg/kg pooled human immunoglobulin to a pooled human IgG administered to the Subject contacts the subject in need thereof daily. In other embodiments, the upper third of the nasal cavity of the subject. methods provided herein for the treatment of Parkinson's In one embodiment of any of the methods provided above, disease include intranasally administering a gel, cream, or the CNS disorder is a neurodegenerative disorder of the ointment composition of pooled human IgG in a dosage/ 45 central nervous system. In a specific embodiment, the neu frequency combination selected from variations 1 to 816 rodegenerative disorder of the central nervous system is found in Table 1 and Table 2. In a particular embodiment, the Alzheimer's disease. In a specific embodiment, the neuro method comprises administering the gel, cream, or ointment degenerative disorder of the central nervous system is Par composition directly to a nasal epithelium of the Subject. In kinson's disease. a particular embodiment, the method comprises administer 50 In one embodiment of any of the methods provided above, ing the gel, cream, or ointment composition directly to the the CNS disorder is a systemic atrophy primarily affecting olfactory epithelium of the subject. the central nervous system. In a specific embodiment, the Specific Embodiments systemic atrophy primarily affecting the central nervous In a first aspect, the disclosure provides a method for system is amyotrophic lateral Sclerosis (ALS). In a specific treating a central nervous system (CNS) disorder in a subject 55 embodiment, the systemic atrophy primarily affecting the in need thereof, the method comprising: delivering a thera central nervous system is Huntington's disease. peutically effective amount of a composition comprising In one embodiment of any of the methods provided above, pooled human immunoglobulin G (IgG) to the brain of the the CNS disorder is an extrapyramidal and movement dis Subject, wherein delivering the composition to the brain order. comprises intranasally administering the composition 60 In one embodiment of any of the methods provided above, directly to a nasal epithelium of the subject. the CNS disorder is a demyelinating disorder of the central In one embodiment of the first aspect, at least 40% of the nervous system. In a specific embodiment, the demylelinat pooled human IgG administered to the Subject contacts the ing disorder of the central nervous system is multiple nasal epithelium of the subject. Sclerosis. In one embodiment of the first aspect, at least 50% of the 65 In one embodiment of any of the methods provided above, pooled human IgG administered to the Subject contacts the the CNS disorder is an episodic or paroxysmal disorder of nasal epithelium of the subject. the central nervous system. US 9,556.260 B2 61 62 In one embodiment of any of the methods provided above, another specific embodiment, the pH of the composition is the CNS disorder is a paralytic syndrome of the central from 4.0 to 6.0. In another specific embodiment, the pH of nervous system. In a specific embodiment, the CNS disorder the composition is from 6.0 to 7.5. is a paralytic syndrome of the central nervous system is In one embodiment of any of the methods provided above, cerebral palsy the composition comprising pooled human IgG is a dry In one embodiment of any of the methods provided above, powder composition. In one embodiment, the dry powder the CNS disorder is a nerve, nerve root, or plexus disorder composition is prepared from an aqueous solution compris of the central nervous system. ing: from 10 mg/mL to 250 mg/mL pooled human IgG, and In one embodiment of any of the methods provided above, from 50 mM to 500 mM glycine. In a specific embodiment, the CNS disorder is an organic mental disorder. 10 the dry powder composition is prepared from an aqueous In one embodiment of any of the methods provided above, solution having a pH of from 4.0 to 7.5. In another specific the CNS disorder is a mental or behavioral disorder caused embodiment, the pH of the composition is from 4.0 to 6.0. by psychoactive Substance use. In another specific embodiment, the pH of the composition In one embodiment of any of the methods provided above, is from 6.0 to 7.5. the CNS disorder is a schizophrenia, schizotypal, or delu 15 In one embodiment of any of the methods provided above, sional disorder. In a specific embodiment, the schizophrenia, the method includes intranasally administering to the Subject Schizotypal, or delusional disorder is Schizophrenia. a dose of from 0.08 mg to 100 mg pooled human IgG per kg In one embodiment of any of the methods provided above, body weight of the Subject (mg IgG/kg). the CNS disorder is a mood (affective) disorder. In a specific In one embodiment of any of the methods provided above, embodiment, the mood (affective) disorder is bipolar disor the method includes intranasally administering to the Subject der. a dose of from 0.2 mg to 40 mg pooled human IgG per kg In one embodiment of any of the methods provided above, body weight of the Subject (mg IgG/kg). the CNS disorder is a neurotic, stress-related, or somatoform In one embodiment of any of the methods provided above, disorder. the method includes intranasally administering to the Subject In one embodiment of any of the methods provided above, 25 a dose of from 0.5 mg to 20 mg pooled human IgG per kg the CNS disorder is a behavioral syndrome. body weight of the Subject (mg IgG/kg). In one embodiment of any of the methods provided above, In one embodiment of any of the methods provided above, the CNS disorder is an adult personality or behavior disor the method includes intranasally administering to the Subject der. a dose of from 0.5 mg to 10 mg pooled human IgG per kg In one embodiment of any of the methods provided above, 30 body weight of the Subject (mg IgG/kg). the CNS disorder is a psychological development disorder. In one embodiment of any of the methods provided above, In one embodiment of any of the methods provided above, the method includes intranasally administering to the subject the CNS disorder is a child onset behavioral or emotional a dose of from 1 mg to 5 mg pooled human IgG per kg body disorder. In a specific embodiment, the child onset behav weight of the Subject (mg IgG/kg). ioral or emotional disorder is Pediatric acute-onset neuro 35 In one embodiment of any of the methods provided above, pyschiatric syndrome (PANS). In another specific embodi the method includes intranasally administering to the Subject ment, the child onset behavioral or emotional disorder is a fixed dose of from 50 mg to 10 g pooled human IgG. Pediatric Autoimmune Neuropsychiatric Disorders Associ In one embodiment of any of the methods provided above, ated with Streptococcal infections (PANDAS). the method includes intranasally administering to the Subject In one embodiment of any of the methods provided above, 40 a fixed dose of from 100 mg to 5g pooled human IgG. intranasal administration of the composition comprises the In one embodiment of any of the methods provided above, use of a non-invasive intranasal delivery device. the method includes intranasally administering to the Subject In one embodiment of any of the methods provided above, a fixed dose of from 500 mg to 2.5g pooled human IgG. intranasal administration of the composition comprises In one embodiment of any of the methods provided above, administration of a liquid drop of the composition directly 45 the method includes intranasally administering to the Subject onto the nasal epithelium. a dose of pooled human IgG at least twice monthly. In one embodiment of any of the methods provided above, In one embodiment of any of the methods provided above, intranasal administration of the composition comprises the method includes intranasally administering to the Subject directed administration of an aerosol of the composition to a dose of pooled human IgG at least once weekly. the nasal epithelium. In a specific embodiment, the aerosol 50 In one embodiment of any of the methods provided above, of the composition is a liquid aerosol. In a specific embodi the method includes intranasally administering to the Subject ment, the aerosol of the composition is a powder aerosol. a dose of pooled human IgG at least twice weekly. In one embodiment of any of the methods provided above, In one embodiment of any of the methods provided above, the composition comprising pooled human IgG does not the method includes intranasally administering to the Subject contain a permeability enhancer. 55 a dose of pooled human IgG at least once daily. In one embodiment of any of the methods provided above, In one embodiment of any of the methods provided above, the composition comprising pooled human IgG consists the method includes intranasally administering to the Subject essentially of pooled human IgG and an amino acid. In a a dose of pooled human IgG at least twice daily. specific embodiment, the amino acid is glycine. In another In one embodiment of any of the methods provided above, specific embodiment, the amino acid is histidine. In another 60 the composition comprising pooled human IgG comprises at specific embodiment, the amino acid is proline. least 0.1% anti-amyloid B IgG. In one embodiment of any of the methods provided above, In one embodiment of any of the methods provided above, the composition comprising pooled human IgG is an aque the method includes administering a second therapy for the ous composition. In one embodiment, the composition com CNS disorder to the subject in need thereof. In one embodi prises: from 10 mg/mL to 250 mg/mL pooled human IgG; 65 ment, the second therapy for the CNS disorder is a cholin and from 50 mM to 500 mM glycine. In a specific embodi esterase inhibitor. In a specific embodiment, the cholinest ment, the pH of the composition is from 4.0 to 7.5. In erase inhibitor is donepezil. In another specific embodiment, US 9,556.260 B2 63 64 the cholinesterase inhibitor is rivastigmine. In another spe TABLE 3-continued cific embodiment, the cholinesterase inhibitor is galan Intranasal administration of IgG to 8 rats to test for intranasaltolerability. tamine. In another specific embodiment, the cholinesterase # Drops Time to inhibitor is tacrine. In another embodiment, the second Rat Weight (g) Drug? Dose delivered perfusion therapy for the CNS disorder is an inhibitor of NMDA-type 3 309.14 Liquid protein 8 (a) 6 L/drop 60 min glutamate receptor. In a specific embodiment, the inhibitor solution—200 (60 IL total) of NMDA-type glutamate receptor is memantine. mg/mL 4 3.09.00 Liquid protein 10 (a) 6 L/drop 60 min EXAMPLES solution—100 (60 IL total) 10 mg/mL 5 342.62 Microsphere—200 10 (a) 6 L/drop 60 min Example 1 mg/mL (60 IL total) 6 355.1 Microsphere—150 10 (a) 6 L/drop 60 min mg/mL (60 IL total) Tolerability of Intranasal Administration of IgG in 7 364.28 Microsphere—200 10 (a) 6 L/drop 60 min Rats 15 mg/mL (60 IL total) 8 348.93 Microsphere—150 28 (a) 6 L/drop 60 min A study was conducted to examine the tolerability of mg/mL (162 L total) intranasal administration of IgG in rats. The purpose of this study was to determine the tolerability of rats to intranasal Results. Three rats received the liquid preparation of IgG administration at various concentrations and prepara intranasal IgG. One rat received 60 uL at 100 mg/ml and it tions. was well tolerated. Two rats received 60 L at 200 mg/mL. Experimental Design: IgG was prepared as a liquid pro The first rat had some difficulty breathing, most likely due tein solution or as a microsphere preparation. The liquid IgG to a problem with light anesthesia. The second rat had some protein Solution was prepared in glycine at 200 mg/mL and difficulties breathing, but survived. Tracheotomies were not 100 mg/mL and had a pH of 5.1-5.3. The IgG microsphere 25 necessary. preparation was prepared at 200 mg/mL and 150 mg/mL in Four rats received the microsphere preparation. Two rats PEG. The IgG preparations were administered to 8 anesthe received 60 uL at 150 mg/ml. One rat received 60 uL at 200 tized, adult male Sprague Dawley rats. mg/ml. One rat received 162 uL at 150 mg/ml. These rats Prior to anesthesia, each rat was weighed. An anesthesia tolerated the highest concentration available at 200 mg/ml cocktail was prepared and full, half, and quarter anesthesia 30 very well. doses were calculated according to the animals weight with The rats tolerated the liquid and microsphere prepara a full dose containing 30 mg/kg ketamine, 6 mg/kg xylazine, tions; however, the rats did tolerate the microsphere prepa and 1 mg/kg acepromazine. The anesthesia was adminis ration better than the protein preparation. tered subcutaneously into the left hind leg, above the thigh. Example 2 Anesthesia was monitored throughout the procedures by 35 assessing reflexes using pinching of the hind paw or tail. If a reflex was present, a half or quarter dose booster was Comparison of Liquid, Microsphere, and Fragment administered as necessary. During drug administration, ani Biodistribution at 30 and 90 Minutes mals received a half dose booster roughly 20-25 min after initial dose if needed. 40 The purpose of this study was to quantify the amount of Anesthetized rats were placed on their backs on a heating intranasally administered IgG that reaches the central ner pad in a metal Surgical tray. The heating pad was connected Vous system and peripheral tissues in anesthetized rats. to a thermostat and was automatically regulated to maintain Specifically, the biodistribution of different formulations and modes of administration were compared. The different for a 37° C. temperature based on continuous measurement mulations and modes of administration are described in from a rectal probe. A 2"x2" gauze pad was rolled tightly 45 into a pillow, taped together, and under the neck to maintain Table 4. a correct neck position horizontal with the counter. A 6LL drop was loaded into a pipette and wiped dry with TABLE 4 a tissue. A cotton Swab covered in parafilm was used to Formulations and modes of administration used in biodistribution study. occlude one naris completely (the flat part of the swab was 50 pushed gently against the naris to prevent airflow), while the *I radiolabeled IgG 6 uL drop was expelled slowly from the pipette (held at a 45° Formulation Mode of Administration angle from the rat’s midline), forming a drop on the pipette Liquid protein formulation Intranasal (biodistribution measured at 30 tip. The drop was lowered onto the open naris to be inhaled. min post administration) The IgG preparations were administered intranasally as 55 Liquid protein formulation Intravenous biodistribution measured at 30 described in Table 3. min post administration) Liquid protein formulation Intranasal (biodistribution measured at 90 min post administration) TABLE 3 Microsphere formulation Intranasal (biodistribution measured at 30 min post administration) Intranasal administration of IgG to 8 rats to test for intranasaltolerability. 60 Microsphere formulation Intranasal (biodistribution measured at 90 # Drops Time to min post administration) Rat Weight (g) Drug? Dose delivered perfusion Microsphere formulation Intranasal (biodistribution measured at 30 (low Ci) min post administration) 1 259.87 Liquid protein 10 (a) 6 L/drop 23 min Antibody fragment (FAb) Intranasal (biodistribution measured at 30 solution—200 (60 IL total) min post administration) mg/mL 2 272.61 Microsphere—50 10 (a) 6 L/drop 60 min 65 mg/mL (60 IL total) Experimental Design: 40 male Sprague-Dawley rats were given one of three preparations of 'I radiolabeled IgG. US 9,556.260 B2 65 66 These included liquid IgG protein solution in glycine at pH A 1 mm transverse incision was made in the femoral vein 5.1-5.3, IgG in a microsphere preparation including PEG, or and a blunted 25 G butterfly needle connected to tubing as Fab antibody fragments in phosphate buffered saline previously filled with 0.9% NaCl and attached to a 3-way (PBS). Drug administration was either intranasal or intra stopcock was immediately inserted. The medial Suture was venous. Rats were sacrificed either 30 or 90 min after the 5 tied down around the needle to secure it in place. To confirm onset of delivery of the IgG preparations for biodistribution placement within the vein, a small amount of blood was studies. withdrawn then saline was pushed. Free Suture strings were For intranasal delivery, the rats were anesthetized and tied to the butterfly needle securing the cannula in place. placed on their backs on a heating pad in a metal Surgical Muscles were protracted, Sutures securing the limbs tray. The heating pad was connected to a thermostat and was 10 automatically regulated to maintain a 37° C. temperature removed, and the Surgical area was covered with gauze wet based on continuous measurement from a rectal probe. A with saline. 2"x2" gauze pad was rolled tightly into a pillow, taped For the intravenous infusion of 'I IgG, a syringe pump together, and under the neck to maintain a correct neck was placed in the hood behind the lead shield. Parts of the position horizontal with the counter. A lead impregnated 15 pump were covered with parafilm (or Saran wrap) to prevent shield was placed between the Surgical tray and the experi contamination with radiation. The pump was set for 4.75 menter for protection against radiation. The dose solution, mm diameter and rate of 50 uL/min. The dose solution (48 pipette, pipette tips, and waste receptacle were arranged uL) was mixed with 452 uL of saline (0.9% NaCl, total behind the shield for easy access. volume 500 mL) in a 1.5 mL microcentrifuge tube. A 1 cc A 6L drop was loaded into the pipette behind the shield Syringe filled with Saline was attached to the 3-way stopcock and wiped dry with a tissue. A cotton Swab covered in attached to the butterfly needle and placed in the pump. A parafilm was used to occlude one naris completely (the flat piece of parafilm was used to secure the Saline Syringe to the part of the Swab was pushed gently against the naris to stopcock. With the stopcock closed to the rat, the pump was prevent airflow), while the 6 ul, drop was expelled slowly started to fill the stopcock with saline. from the pipette (held at a 45° angle from the rat’s midline), 25 A 1 cc syringe attached to a 27G or 30G needle was used forming a drop on the pipette tip. The drop was lowered onto to collect the drug from the microcentrifuge tube and then the open naris to be inhaled. After two minutes, the alternate the Syringe was connected to the 3-way stopcock. The naris was occluded and a 6 LL drop was administered in the stopcock was turned so that the flow was open between the same fashion. A drop was administered as described above dose solution and the rat. The tubing was filled with dose every two minutes to alternating nares until a total of8 drops 30 Solution making Sure that no air bubbles are pushed into the was delivered (4 to each naris) over 14 min. Delivered time rat and that fluid does not pool near the femoral vein (this of each drop was noted as well as any details regarding the would indicate the needle was not in the vein). The stopcock animal's respiration or success of the delivery. Three 3 ul. was turned so that flow was open to the Saline Syringe and aliquots of each dosing Solution were gamma counted to the rats. determine the measured specific activity. 35 The time and start Volume of the saline Syringe was noted For intravenous IgG delivery, the rats required cannula and the pump was started. The stop Volume of the saline tion of the femoral artery. Anesthetized animals were posi Syringe was also noted at the end of the 14 min infusion. At tioned on their backs in Surgical tray on a heating pad least 700 uL of saline was infused (50 uL/min over 14 min). maintained at 37° C. Both hind legs were secured by loosely The volume of saline administered was slightly more than tying a Suture around the limbs and weighting them with a 40 the volume of the tubing which ensured that all of the dose hemostat. Small, superficial cuts with blunt scissors were Solution was administered. made at the mid inguinal point, making Sure not to cut the Two minutes prior to the desired end point time, anes Superficial blood vessels. Gentle, blunt dissection using thetized animals were laid flat on their backs in a metal cotton Swabs exposed the femoral vein from the great Surgical tray. The heating pad, rectal probe, and neck pillow Saphenous vein to the inguinal ligament. Blunt Scissors were 45 were removed. Tape was used to secure the front limbs to the used to cut away the skin to get a better view the area. pan. The back of the pan was elevated slightly to allow blood Overlying muscle was retracted by threading a 4-0 Suture to run away from the animal. The sternum was exposed by with a curved needle through the muscle, attaching a curved cutting through the skin. The sternum was clamped with a hemostat to the end of the Suture and weighting it in place. hemostat and the rib cage was cut open laterally, exposing Connective tissue Surrounding the femoral vein and artery 50 the diaphragm. The diaphragm was cut laterally to expose was carefully removed with blunt dissection (cotton swabs). the pleural cavity. Connective tissue between the vein and artery was teased Surgical Scissors were used to cut up the sides of the apart using two pairs of forceps carefully using a motion ribcage toward the armpits of the animal, creating a 'V' running parallel to the blood vessels and being careful not to shaped incision exposing the heart. The hemostat holding rupture the vessels. Saline was applied if the area was dry. 55 the sternum was taped above the head to hold the cavity In an area free of branches, the angled forceps was open. The heart was stabilized using the blunt forceps while inserted underneath the vein, the tip poked through the a small cut was made into the left ventricle. A 1 cc-Syringe connective tissue, and the forceps slowly opened to pull a 12 with 18 G, 1" blunt needle was inserted into the left ventricle inch 4-0 suture through very carefully. If the vein collapsed, and approximately 0.1 mL of blood was removed and placed a cotton Swab was used to gently pump the vein full of 60 into a pre-weighed tube for gamma counting. A second 18 G blood. A second Suture was pulled through in a similar blunt needle attached to an extension set filled with 60 cc of manner. The medial and lateral sutures were tied into loose saline was inserted through the left ventricle and into the knots. A cotton swab was used to pump the vein full of . A large bulldog clamp was placed just above the heart blood. The lateral suture (closest to the knee) was tied into on the aorta, securing the blunt needle in place. a tight knot. A hemostat was attached to the Suture strings of 65 The animal was perfused with 60 mL of saline followed the medial Suture and some tension was added to occlude by 360 mL of paraformaldehyde using a syringe pump at a blood flow. rate of 15 mL/min. US 9,556.260 B2 67 68 Throughout experimental procedures, strict precautions For body dissection, bodies were placed on their backs were followed to prevent radioactive contamination of ani and a longitudinal cut using a scalpel was used to open the mal tissues, Surgical tools, and equipment. Geiger counters peritoneal cavity down to the bladder. 3 mm square samples were placed at each work station to continuously screen of liver (superficial right lobe), kidney (left, tip), renal artery, tools, workspace, and staff. Personal protective equipment 5 spleen (tip), lung (right, top lobe), and heart were collected. including double layered gloves, lab coats, eye protection, Approximately 0.1-0.2 mL of urine was collected. masks, and bouffant caps were worn at all times. Lead Bodies were flipped over onto the stomach and a super impregnated shields were used to minimize exposure to ficial incision was made down the length of the animal from radiation. Radioactive monitoring badges were also worn by shoulders to hips, following the spine. The skin was peeled staff throughout experimental procedures to quantify expo 10 away from the underlying tissue on both sides to expose the SUC. Immediately after collection, each tissue sample was shoulder blades. Axillary nodes in the armpits were dis placed into a pre-labeled and pre-weighed gamma tube for sected and cleared of connective tissue. A piece of right later measurement. deltoid muscle was collected (-3 mm). For brain dissection, skin and muscle around the neck 15 The muscles overlying the spine were scored with a were cut with a scalpel just above the shoulder blades and a Scalpel. To expose the spinal cord, a small hemostat was large pair of Scissors used to decapitate the animal, cutting inserted into the spinal column and used to chip away dorsal to Ventral to avoid contamination from the trachea overlying vertebrae and tissues. A Small spatula was used to and esophagus. To expose the brain, a midline incision was loosen the cord from the spinal cavity and forceps used to made on the dorsal side of the skull, then skin was peeled remove it and place into a petri dish. The dura was peeled off away, and a straight hemostat was used to break the bone, of the cord using forceps. The cord was dissected into lower taking care to leave the dorsal dura attached. Dorsal dura cervical, thoracic, and lumbar portions. The top ~2 mm of was collected. lower cervical segment was discarded. To remove the brain from the skull, the head was inverted A 2 cm segment of trachea and esophagus was dissected and a small spatula was used to free it from the cavity. The 25 from the body and connective tissues were removed. The top posterior optic nerve and trigeminal nerves were cut close to 0.5 cm (closest to the decapitation point) of each was the brain. The brain was then placed into a clean Petri dish discarded. for dissection. Pre-weighed gamma tubes containing samples were From the base of the skull, the ventral dura was collected reweighed to determine tissue weight. Tissue samples from by Scraping a forceps on the Ventral skull walls. The 30 the rats were counted using a COBRA II Auto-Gamma pituitary, optic chiasm, and trigeminal nerves were col Counter using a standard I protocol and a 5 min count lected. The anterior portion of the trigeminal nerve consisted time. Counters were normalized weekly to ensure a counting of the portion before the visible branch in the skull, while the efficiency at or above 80%. Background counts were sub remainder containing the trigeminal ganglion was consid tracted. ered as the posterior section. The head was then set aside and 35 covered with a kim-wipe for later dissection. Mean and standard error of the nM concentration of each A microscope was used to help remove vessels from the tissue sample were calculated. Any value outside two stan brain. Using Surgical forceps, microscissors, and a 30 G dard deviations of the mean for each tissue was considered needle, the basilar artery and circle of Willis were removed an outlier and removed from the data set. nM IgG concen and placed onto pre-weighed paper (paper was used because 40 trations were calculated for each tissue using the measured of the small weight of this tissue). The needle was used to specific activity of dosing solutions, the CPM of each tissue, lift the vessels away from the brain, the forceps to grab hold, and the Volume of each tissue (assuming 1 g 1 mL). and the microscissors to make the cuts. This tissue was Results, Intranasal IgG Liquid Preparation Distribution at weighed immediately upon collection and then the entire 30 min End Point. Eight rats received IN IgG liquid prepa paper was crumpled and placed into the bottom of tube). 45 ration at an average dose of 6.0 mg in 47.4 uL containing Prior to placing the brain into the coronal matrix, the 69.6 uCi with a 30 min end point. Animals tolerated the IN olfactory bulbs were cut off at the natural angle using a razor administration well and all survived until the 30 min desired blade. In the coronal brain matrix, a razorblade was inserted end point. at the center of where optic chiasm was before removal to At the site of IN drug administration, the average IgG normalize each animal to the same location (bregma). Addi 50 concentrations in the respiratory and olfactory epithelia were tional blades were placed every 2 mm from the first blade, 136.213 nM and 442 nM respectively. A rostral to caudal resulting in 6x2 mm slices, 3 rostral to the optic chiasm and gradient of 13.1 nM to 6.0 nM. IgG was observed in the 3 caudal. trigeminal nerve. A similar gradient from the olfactory bulb Blades were removed and tissues were dissected from to the anterior olfactory nucleus of 4.1 nM to 1.5 nM IgG each slice (FIG. 1A-1F). The remaining section of cortex 55 was observed. The average cortex concentration of IgG after and hippocampus was dissected from the remaining brain IN administration was 1.3 nM. Concentrations of IgG in tissue in the matrix and placed in respective tubes. The upper other brain regions ranged from a low of 0.7 nM in the cervical spinal cord was collected. The remaining brain was striatum to a high of 1.7 nM in the hypothalamus. The then bisected along the midline and dissected into midbrain, hippocampus was found to contain 0.6 nM IgG. A rostral to pons, medulla, and cerebellum according to FIG. 1G. 60 caudal concentration gradient (1.6 nM to 0.7 nM) was Returning to the head, the ventral side of the neck was cut observed within the extra brain material sampled. Similarly, anteriorly and skin peeled back exposing lymph nodes, a rostral to caudal concentration gradient (1.2 nM to 0.3 nM) salivary glands, and neck muscles. The Superficial nodes, was observed in the spinal cord. The average concentration deep cervical nodes, carotid , and thyroid gland were of IgG in the dura of the brain was 15.2 nM compared to a dissected and cleared of connective tissue. A razorblade was 65 spinal cord dura concentration of 2.8 nM. The dura likely used to bisect the skull along the midline. The olfactory also contains some or most of the arachnoid membrane and epithelium and respiratory epithelium were collected. together comprise two of the three components of the US 9,556.260 B2 69 70 meninges. Other tissues sampled from the cavity of the Results, Intranasal IgG Microsphere Preparation (low ventral skull (pituitary and optic chiasm) contained 8.2 nM uCi) Distribution at 30 min End Point. Four rats received IN and 7.4 nM IgG respectively. IgG microsphere preparation (low uCi) at an average dose of The blood concentration of IgG at the 30 min end point was 13.9 nM. Concentrations of IgG in peripheral organs 7.2 mg in 48.0 LA containing 24.7 uCi with a 30 min end ranged from a low of 1.3 nM in the heart to a high of 6.1 nM point. The raw data from the four rats is provided in Table in the spleen and kidney, with urine containing 8.1 nM. 5. The measured specific activity from this dosing Solution Concentrations of IgG in the basilar and carotid arteries was much lower than expected based upon the provided were considerably higher than the renal artery (11.7 and 14.1 nM versus 4.4 nM). Average concentration of IgG in the specific activity. Animals tolerated the IN administration sampled lymph nodes was 4.7 nM. Levels of IgG in tissues 10 well and all survived until the 30 min desired end point. Zero measured to assess variability of IN administration and statistically significant outliers and fourteen non-statistically breathing difficulty (lung, esophagus, and trachea) were significant outliers were identified out of a total of 211 consistent across animals. concentration values. TABLE 5 Biodistribution (nM concentrations) of intranasally administered IgG microsphere preparations (with low uCi) at the 30 min end point with outliers included. BAX-17 BAX-18 BAX-19 BAX-20 Avg SE Volume Delivered (L) 48.0 48.0 48.0 48.0 48.0 O.OO uCi Delivered 20.9 20.9 28.6 28.6 24.7 +2.2 mg Delivered 7.2 7.2 7.2 7.2 7.2 O.OO Olfactory Epithelium 6,806.0 3,931.1 15,573.6 2O3.9 6,628.6 +3,273.6 Respiratory Epithelium 559,241.5 268,256.5 219,595.4 25,412.0 268,126.3 +110,307.7 Anterior Trigeminal 9.7 28.6 11.4 4.6 13.6 5.2 Nerve Posterior Trigeminal 5.4 14.5 6.3 4.1 7.6 +2.4 Nerve Olfactory Bulbs 5.9 3.4 3.7 4.1 4.3 O.6 Anterior Olfactory 1.9 2.4 2.1 1.4 1.9 O.2 Nucleus Frontal Cortex 1.2 1.6 2.O 1.4 1.6 O.2 Parietal Cortex O.8 1.2 1.1 O.S O.9 O.2 Temporal Cortex O.9 : 1.2 O.6 O.9 0.2 Occipital Cortex O.O 1.3 0.4 1.6 O.8 +0.4 Extra Cortex 1.6 1.4 1.1 O.9 1.3 O.2 Amygdala 1.6 S.1 1.9 0.7 2.3 O.9 Striatum O.8 16.5 O.6 O6 4.6 +4.0 Septal Nucleus 1.8 4.2 O.6 O.3 1.7 O.9 Hypothalamus 1.8 3.9 2.5 1.1 2.3 O.6 Thalamus O.3 O.9 O.6 0.4 O.S O. Midbrain O.8 1.6 0.7 O6 O.9 O.2 Hippocampus O6 1.3 0.7 0.4 0.7 O.2 Pons O6 1.9 1.2 O.8 1.1 O.3 Medulla 0.7 1.2 1.1 O.8 1.O O. Cerebellum O6 1.2 O.8 O6 O.8 O.2 Extra Slice #1 1.3 2.6 2.4 1.5 2.0 O.3 Extra Slice #2 1.O 1.1 1.2 1.1 1.1 O.05 Extra Slice #3 0.7 1.1 1.O 0.7 O.9 O. Extra Slice #4 0.7 1.2 O.8 O6 O.8 O. Extra Slice #5 O6 1.O O.8 O.S 0.7 O. Extra Slice #6 0.7 1.3 1.O O6 O.9 O.2 Pituitary 7.0 18.1 6.2 3.6 8.7 3.2 Optic Chiasm 14.9 19.0 8.2 8.1 12.5 2.7 Dorsal Dura 12.O 20.7 15.1 2O6 17.1 +2. Ventral Dura 18.5 56.5 16.2 15.2 26.6 10.0 Spinal Dura 2.9 1.O 1.7 3.6 2.3 O.6 Upper Cervical Spinal 1.O 1.2 1.O 1.5 1.2 O. Cord Lower Cervical Spinal 0.4 O.3 0.4 O.9 O.S O. Cord Thoracic Spinal Cord O.S 0.4 O.6 0.7 O.S O. Lumbar Spinal Cord O2 O.2 O.2 O.3 O.2 O.O3 Circle of Willis & Basilar 24.9 29.7 17.7 9.3 20.4 +4.4 Artery Carotid Artery 2O7.3 17.9 14.1 13.1 63.1 48. Renal artery (L) 6.1 2.4 4.5 2.4 3.8 O.9 Superficial Nodes (2) 30.8 17.1 6.9 O.8 13.9 6.6 Cervical Nodes (2) 7.7 3.4 9.2 62.7 20.8 +14.0 Axillary Nodes (2) 4.2 2.1 2.2 2.8 2.8 O.S Blood Sample 2,889.3 8.0 1,730.1 7.1 1,158.6 705.4 Muscle (R, deltoid) 3.7 2.1 1.2 1.4 2.1 O.6 Liver (R, Superficial lobe) 1.2 1.O O.9 O.9 1.O O.1 Kidney (L, tip) 13.1 2.5 6.9 3.0 6.4 2.5 Orine 5.4 4.0 9.3 3.0 5.4 +1.4 Spleen (tip) 3.1 1.2 3.0 1.8 2.3 O.S US 9,556.260 B2 71 72 TABLE 5-continued Biodistribution (nM concentrations) of intranasally administered IgG microsphere preparations (with low uCi) at the 30 min end point with outliers included. BAX-17 BAX-18 BAX-19 BAX-2O Avg SE

Heart 4.4 2.9 0.4 0.7 2.1 O.9 Lung (R, top lobe) 3.4 5.2 2.3 2.6 3.4 O.6 Thyroid 28,623.9 102.6 30,320.6 15.7 14,765.7 +8.497.9 Esophagus 3.7 2.5 2.7 4.7 3.4 O.S Trachea 2.7 1.5 2.O 4.9 2.8 O.8 Drug Standard CPM 2,316,335 2,316,335 3,256,120 3,256,120 2,786.228 +271,292.6 Drug Standard CPM 2,380,434 2,380.434 3,216.298 3,216.298 2,798,366 +241,293.2 Drug Standard CPM 2,259,775 2,259,775 3,051.466 3,051.466 2,655,621 +228,541.5 * = negative tube weight, so nM could not be calculated 15 Results, Intranasal IgG Microsphere Preparation Distri- data from the eight rats is provided in Table 6. Animals bution at 30 min End Point. Eight rats received IN IgG microsphere preparation at an average dose of 7.2 mg in 48.0 tolerated the IN administration well and all survived until uL containing 60.0 uCi with a 30 min end point. The raw the 30 min desired end point. TABLE 6 Biodistribution (nM concentrations) of intranasally administered IgG microsphere preparations at the 30 min end point with outliers excluded. BAX-21 BAX-22 BAX-23 BAX-25 BAX-26 BAX-28 Avg SE

Volume Delivered 48.0 48.0 48.0 48.0 48.0 48.0 48.0 O.OO (LL) uCi Delivered 59.9 56.3 73.4 60.8 53.1 56.5 6O.O 2.9 mg Delivered 7.2 7.2 7.2 7.2 7.2 7.2 7.2 O.OO Olfactory Epithelium X 377.5 629.1 X 97.9 2010 326.4 116.3 Respiratory 23,108.2 20,219.7 33,657.6 87,547.5 183,182.6 101,353.0 74,844.8 +25,792.8 Epithelium Anterior Trigeminal 2.O 1.7 2.1 1.3 O.8 1.2 1.5 O.2 Nerve Posterior Trigeminal 2.O 1.2 1.3 O.8 0.7 O.9 1.1 O.2 Nerve Olfactory Bulbs 2.7 1.O 1.1 O6 1.O 0.7 1.2 O.3 Anterior Olfactory 0.7 1.2 0.4 0.4 O.3 O.3 O6 O.1 Nucleus Frontal Cortex O.8 O.3 O.8 0.4 0.4 0.4 O.S O.1 Parietal Cortex O.3 O6 O.6 O.2 0.4 0.4 0.4 O.1 Temporal Cortex O.2 0.4 O.2 1.3 O.2 O.3 O.S O.2 Occipital Cortex O.3 O.1 0.4 0.4 2.3 O6 0.7 O.3 Extra Cortex O.3 O.3 O.9 O.3 O.2 O.3 0.4 O.1 Amygdala O.2 X O.2 0.4 O.3 O.3 O.3 O.04 Striatum O.6 1.5 O.3 1.1 X 2.1 1.1 O.3 Septal Nucleus 0.4 0.7 O.1 1.1 O6 O6 O6 O.1 Hypothalamus 0.7 O.S O.3 0.4 O.3 O6 O.S O.1 Thalamus O.1 O.1 O.2 O.1 O.1 O.1 O.1 O.O1 Midbrain O.2 O.2 O.S O.2 O.2 O.2 O.3 O.OS Hippocampus O.2 O.3 O.2 O.2 O.1 O.1 O.2 O.O3 Pons 0.4 O.3 O.S O.3 O.S O.3 0.4 O.04 Medulla O.3 O.2 0.4 O.3 O.2 O.2 O.3 O.04 Cerebellum O.3 1.1 X 1.7 O.2 O.2 0.7 O.3 Extra Slice #1 1.1 O.3 0.4 0.4 O.3 0.4 O.S O.1 Extra Slice #2 O.6 O.2 O.2 O.2 O.2 2.4 O6 +0.4 Extra Slice #3 0.4 O.3 O.3 O.2 O.2 O.S O.3 O.04 Extra Slice #4 O.3 O.2 O.2 O.2 O.1 2.8 O6 +0.4 Extra Slice #5 O.2 O.2 O.3 O.2 O.2 O.2 O.2 O.O2 Extra Slice #6 O.2 O.2 O.2 O.2 O.2 O.2 O.2 O.O1 Pituitary 1.8 1.3 3.2 2.9 1.7 2.6 2.2 O.3 Optic Chiasm 2.2 1.4 2.5 2.1 1.4 0.7 1.7 O.3 Dorsal Dura 5.4 7.8 S.O 2.4 S.1 1.6 4.6 O.9 Ventral Dura 9.4 3.1 2.9 3.7 3.1 1.7 4.0 +1.1 Spinal Dura O.6 0.4 O.8 O.2 X O6 O.S O.1 Upper Cervical O.SO O.36 O.S2 O.28 O.34 O.S3 O42 O.04 Spinal Cord Lower Cervical O.O6 O.14 O.13 O.10 O.12 O.12 O.11 O.O1 Spinal Cord Thoracic Spinal O.O6 O.O3 O.08 O.09 X O.04 O.O6 O.O Cord Lumbar Spinal Cord O.O8 O.O7 O.10 O.OS O.O7 O.O3 O.O6 O.O1 Circle of Willis & 11.5 X 15.7 12.4 2.0 5.2 9.3 2.5 Basilar Artery US 9,556.260 B2 73 74 TABLE 6-continued Biodistribution (nM concentrations) of intranasally administered IgG microsphere preparations at the 30 min end point with outliers excluded. BAX-21 BAX-22 BAX-23 BAX-25 BAX-26 BAX-28 Avg SE Carotid Artery 4.6 5.4 1.6 19 X 1.9 3.1 Renal artery (L) O.9 0.4 O.S 0.7 O.6 O.S O.6 Superficial Nodes O.8 0.7 O.9 X 4.3 O.8 1.5 (2) Cervical Nodes (2) 1.2 1.9 1.1 O.8 X O.S 1.1 O2 Axillary Nodes (2) 0.4 X O.3 OS 1.O O.S O.S O1 Blood Sample 156.7 261.5 1.1 19 362.3 268.7 175.4 61.1 Muscle (R, deltoid) O.1 O.9 O.3 O3 0.7 O.2 0.4 O1 Liver (R, Superficial O.O X O.1 O2 O.3 O.3 O.2 O.OS obe) Kidney (L, tip) O6 O.3 0.4 1.O 1.O 1.2 O.8 O1 Orine O6 1.1 O.9 O.9 3.5 2.7 1.6 OS Spleen (tip) O.3 0.4 O6 O6 0.4 O1 Heart O.3 0.4 O.3 O1 O.1 O.3 O.04 Lung (R, top lobe) O.S 0.4 O.3 2.2 O.2 1.3 O3 Thyroid 1,697.8 3,275.2 16.1 36.2 X 35.6 1O12.2 651.5 Esophagus O6 0.4 O.1 0.7 1.3 0.4 O2 Trachea O.S 1.O O.3 O6 O.8 O6 O.6 O1 Drug Standard 6,936,801 6,170,223 8,071,624 7,024,714 6,006,357 6,587,524 6,799,540.2 +303,198.0 CPM Drug Standard 6,854,563 6,687,656 8,239,126 6,958,531 6,134,932 6,360,075 6,872,480.3 +300,895.5 CPM Drug Standard 6,894,326 6,596,846 9,035,030 7,046,819 6,205,338 6,576,363 7,059,120.2 +412,602.5 CPM X = outlier removed from analysis

At the site of IN drug administration, the average IgG The blood concentration of IgG at the 30 min end point concentrations in the respiratory and olfactory epithelia were 30 was 175 nM. Concentrations of IgG in peripheral organs 74,844.8 nM and 326 nM respectively. A rostral to caudal ranged from a low of 0.2nM in the liver to a high of 0.8 nM gradient of 1.5 nM to 1.1 nM. IgG was observed in the in the kidney, with urine containing 1.6 nM. Concentrations trigeminal nerve. A similar gradient from the olfactory bulb of IgG in the basilar and carotid arteries were considerable to the anterior olfactory nucleus of 1.2 nM to 0.6 nM IgG greater than the concentration in the renal artery (9.3 and 3.1 35 nM versus 0.6 nM). Average concentration of IgG in the was observed. The average cortex concentration of IgG after sampled lymph nodes was 1.0 nM. Levels of IgG in tissues IN administration was 0.5 nM. Concentrations of IgG in measured to assess variability of IN administration and other brain regions ranged from a low of 0.1 nM in the breathing difficulty (lung, esophagus, and trachea) were thalamus to a high of 1.1 nM in the striatum. The hippocam consistent across animals. IgG levels in the thyroid varied pus was found to contain 0.2 nM IgG. The average concen 40 greatly ranging from 16.1 nM to 3,275 nM, even after the tration of IgG in the extra brain material sampled was 0.4 removal of outliers. nM. similar to the average cortex concentration, and a Results, Intranasal IgG Fragment Preparation Distribution concentration gradient was not observed. A rostral to caudal at 30 min End Point. Four rats received an IN IgG Fab concentration gradient (0.42 nM to 0.06 nM) was observed antibody fragment preparation at an average dose of in the spinal cord. The average concentration of IgG in the 45 approximately 3.3 mg in 48.2 LL containing 76.4 uCi. The dura of the brain was 4.3 nM compared to a spinal cord dura raw data from the four rats is provided in Table 7. All four concentration of 0.6 nM. Other tissues sampled from the experiments were completed with an end point of 30 min, Ventral skull, the pituitary and optic chiasm, contained 2.2 and as expected the animals tolerated the IN administration nM and 1.7 nM IgG respectively. well and all survived until the desired end point. TABLE 7 Biodistribution (nM concentrations) of intranasally administered IgG Fab preparations at the 30 min end point with outliers excluded. BAX-41 BAX-42 BAX-43 BAX-44 Avg SE Volume Delivered (IL) 48.1 48.1 48.2 48.2 48.2 O.O uCi Delivered 76.7 76.7 76.O 76.O 76.4 O.2 mg Delivered 3.3 3.3 3.3 3.3 3.3 O.O Olfactory Epithelium 232.4 435.2 271.2 X 312.9 62.1 Respiratory Epithelium 93,166.9 138,501.7 59,830.3 140,806.9 108076.4 +19,465.7 Anterior Trigeminal 72.8 101.4 1415 73.3 97.2 16.2 Nerve Posterior Trigeminal 32.4 34.2 33.9 19.8 30.1 3.4 Nerve Olfactory Bulbs S4.O 26.3 45.2 23.7 37.3 7.3 Anterior Olfactory 20.4 14.1 2S.O 15.9 18.8 +2.4 Nucleus Frontal Cortex 2O.O 11.9 21.5 X 17.8 3.O US 9,556.260 B2 75 76 TABLE 7-continued Biodistribution (nM concentrations) of intranasally administered IgG Fab preparations at the 30 min end point with outliers excluded. BAX-41 BAX-42 BAX-43 BAX-44 Avg SE

Parietal Cortex 7.0 5.8 1.6 6.7 7.8 1.3 Temporal Cortex S.6 4.3 9.5 4.3 5.9 +1.2 Occipital Cortex 9.3 S.6 7.1 10.0 8.O 1.O Extra Cortex 8.9 7.0 8.5 4.2 7.2 +1.1 Amygdala 10.3 14.3 5.2 6.9 11.7 1.9 Striatum S.O S.O 8.6 3.8 S.6 1.O Septal Nucleus 8.4 6.8 O.8 S.1 7.8 +1.2 Hypothalamus 18.0 18.1 22.7 6.3 16.3 3.5 Thalamus S.1 8.2 9.8 2.9 6.5 1.5 Midbrain 8.9 10.3 1.O 4.0 8.6 +1.6 Hippocampus 6.1 7.4 7.2 2.6 5.8 +1.1 Pons 11.O 12.4 2.4 4.9 10.2 1.8 Medulla 11.O 1O.S 1.3 S.O 9.4 1.5 Cerebellum 9.2 5.5 6.1 8.3 7.3 0.9 Extra Slice #1 27.6 16.8 31.2 32.5 27.0 3.6 Extra Slice #2 12.5 9.8 6.O X 12.8 1.8 Extra Slice #3 8.5 8.1 1.5 13.9 1O.S +1.4 Extra Slice #4 7.4 6.5 9.2 6.2 7.3 O.7 Extra Slice #5 6.8 X 8.1 14.3 9.7 2.3 Extra Slice #6 6.O 5.5 7.4 4.1 5.7 O.7 Pituitary 41.6 44.2 SO.6 34.O 42.6 3.4 Optic Chiasm 31.8 21.8 36.4 12.4 25.6 54 Dorsal Dura 138.2 115.3 129.9 101S 121.2 8.1 Ventral Dura 1231 109.7 106.0 81.1 1OS.O 8.8 Spinal Dura 3.4 8.1 2.7 4.5 4.7 +1.2 Upper Cervical Spinal 2O.S 13.7 16.7 7.9 14.7 2.7 Cord Lower Cervical Spinal 1.O 0.7 O.9 1.3 1.O 0.1 Cord Thoracic Spinal Cord O.9 0.7 O.8 1.3 O.9 0.1 Lumbar Spinal Cord 0.7 O.6 O6 O.8 0.7 0.1 Circle of Willis & 64.O 846 69.8 44.4 65.7 8.3 Basilar Artery Carotid Artery X 36.0 35.5 42.9 38.1 +2.4 Renal artery (L) 9.9 14.8 4.0 5.9 8.6 +2.4 Superficial Nodes (2) 9.0 9.4 5.5 6.7 7.6 0.9 Cervical Nodes (2) 19.5 X 23.8 32.O 25.1 3.7 Axillary Nodes (2) 3.2 6.2 3.6 4.1 4.3 O.7 Blood Sample 31.2 38.4 28.9 33.2 32.9 2.0 Muscle (R, deltoid) 2.87 5.05 2.26 2.18 3.1 O.7 Liver (R, Superficial 3.8 3.3 4.0 2.4 3.4 0.3 obe) Kidney (L, tip) 11.1 21.5 4.0 13.1 12.4 3.6 Orine 10.6 10.3 19.9 9.0 12.4 2.5 Spleen (tip) 9.7 12.9 3.4 9.0 8.7 2.0 Heart O.8 3.0 4.5 1.5 2.5 0.8 Lung (R, top lobe) 3.5 9.1 6.7 4.4 5.9 +1.2 Thyroid 228.2 411.7 230.1 273.2 285.8 43.2 Esophagus 4.1 6.4 X 5.8 5.4 O.7 Trachea S.6 8.7 11.3 4.8 7.6 1.5 Drug Standard CPM 7,158,905 7,158,905 6,994.454 6,994.454 7076679.3 +47,472.8 Drug Standard CPM 6,974,631 6,974,631 7.215,418 7.215,418 7095.024.0 +69,509.2 Drug Standard CPM 7,280,104 7,280,104 7,020,805 7,020,805 7150454.3 +74,853.3 X = outlier removed from analysis

At the site of IN drug administration, the average IgG Fab Similarly, a rostral to caudal concentration gradient (14.7 concentrations in the respiratory and olfactory epithelia were nM to 0.7 nM) was observed in the spinal cord. The average concentration of IgG Fab in the dura of the brain was 113.1 108,076 nM and 313 nM respectively. A rostral to caudal 55 nM compared to a spinal cord dura concentration of 4.7 nM. gradient of 97.2 nM to 30.1 nM. IgG Fab was observed in the Other tissues sampled from the cavity of the ventral skull trigeminal nerve. A similar gradient from the olfactory bulb (pituitary and optic chiasm) contained 42.6 nM and 25.6 nM IgG Fab respectively. to the anterior olfactory nucleus of 37.3 nM to 18.8 nM IgG The blood concentration of IgG Fab at the 30 min end Fab was observed. The average cortex concentration of IgG 60 point was 32.9 nM. Concentrations of IgG Fab in peripheral Fab after IN administration was 9.3 nM. Concentrations of organs ranged from a low of 2.5 nM in the heart to a high IgG in other brain regions ranged from a low of 5.6 nM in of 12.4 nM in the kidney and urine, with the spleen con taining 8.7nM. Concentrations of IgG Fab in the basilar and the striatum to a high of 16.3 nM in the hypothalamus. The carotid arteries were considerably higher than the renal hippocampus was found to contain 5.8 nM IgG Fab. A 65 artery (65.7 and 38.1 nM versus 8.6 nM). rostral to caudal concentration gradient (27.0 nM to 5.7 nM) Results, Comparison of 30 min and 90 min end points. was observed within the extra brain material sampled. Concentrations of IgG in brain tissues were generally similar US 9,556.260 B2 77 78 or slightly higher with the extended 90 min end point as (thalamus, midbrain) and some tissues containing much less compared to the 30 min end point for the IgG liquid (striatum, occipital cortex) at the 90 min vs. the 30 min end preparation. There was more variability in the IgG micro- points. Summaries of the IgG concentrations in tissues are sphere preparation, with Some tissues containing much more provided in Table 8 and Table 9. TABLE 8 Summary of tissue concentrations (nM E SE) of IN. IV, and Fab IgG at 30 min and 90 min endpoints with Outliers removed. Treatment IgG Protein (mean nM E SE IgG MicroSpheres (mean nM it SE). IgG FAB (mean nM t SE) Route Intravenous Intranasal Intranasal Intranasal

Time Point 30 min 90 min 30 min 90 min 30 min

Sample Size n = 7 n = 8 n = 6 n = 6 n = 5 n = 4 Volume Delivered (IL) 47.7 O.2 47.4 O2 47.6 O.1 48.O. O.OO 48.O. O.OO 48.2 O.O uCi Delivered 69.5 O.3 69.6 O.3 7O.O. O.O1 60.0 - 29 59.7 2.0 76.4 O2 mg Delivered 6.O. O.O3 6.O. O.O2 7.4 O.OO 7.2 OOO 7.2 O.OO 3.3 O.O Olfactory Epithelium 43.0 - 3.7 441 - 185 3.SS 71 326 116 3,192 + 1,625 312.9 62.1 Respiratory 41.1 + 4.3 136,213 + 163,627 + 74,845 + 124,509 + 108076.4 Epithelium 27,325 16,376 25,793 20,723 19,465.7 Anterior Trigeminal 10.5 - 1.0 13.1 2.6 19.3 2.8 1.5 + 0.2 8.0 - 1.3 97.2 16.2 Nerve Posterior Trigeminal 6.3 1.O 6.O. 11 8.4 1.7 1.1 O2 3.1 O.2 30.1 - 3.4 Nerve Olfactory Bulbs 3.4 O.S 4.1 - 0.9 9.9 1.6 1.2 - 0.3 1.5 + 0.2 37.3 7.3 Anterior Olfactory 1.9 O.3 1.5 + 0.2 2.5 + 0.3 O.6 0.1 O.6 O. 18.8 24 Nucleus Frontal Cortex 2.9 - O.S 1.4 + O. 3.8 - 0.6 O.S. O.1 O.7 O. 17.8 3.0 Parietal Cortex 3.3 O.7 O.9 O. 5 - 0.1 O.4 0.1 O3 + O. 7.8 1.3 Temporal Cortex 2.90.7 1.1 + O. .4 + 0.2 OS O2 O.S. O. 5.9 1.2 Occipital Cortex 2.3 O.2 1.8 O.3 2.5 + 0.2 O.7 O.3 O3 + O. 8.0 - 1.0 Extra Cortex 1.8 O.3 1.O. O. 9 O.2 O.4 0.1 O.S. O. 7.2 - 1.1 Amygdala 1.9 O.1 1.4 + 0.2 6 - 0.2 O.3 0.04 0.4 + 0. 11.7 - 1.9 Striatum 1.8 - 0.2 0.7 - 0. O.9 - 0.1 1.1 - 0.3 0.6 - 0.2 5.6+ 1.0 Septal Nucleus 1.8 O.1 O.9 O. .1 + 0.1 O.6 0.1 O.6 O.4 7.8 1.2 Hypothalamus 2.0 O.2 1.7 O.3 9 O.2 O.S. O.1 O.6 O. 16.3 3.5 Thalamus 1.7 O.3 O4 O.O3 O.6 0.04 O.1 OO1 O3 + O. 6.5 - 1.5 Midbrain 1.8 O.3 O.7 O. 3 - 0.1 O3 + O.OS O.S. O. 8.6 1.6 Hippocampus 1.1 + 0.1 O.6 O. O. O.1 O.2 O.O3 O.S. O. 5.8 1.1 Pons 1.7 O.2 O.9 O. 6 - 0.2 O.4 0.04 O.S. O. 10.2 - 18 Medulla 1.8 O.3 O.9 O. 6 - 0.2 O.3 0.04 O.4 O.04 94 - 1.5 Cerebellum 1.9 O.3 O.8 O. 7 0.2 O.7 O.3 O.S. O. 7.3 O.9 Extra Slice #1 2.0 O.2 1.6 O.3 3.3 0.4 O.S. O.1 O.8 O. 27.036 Extra Slice #2 2.1 O.3 1.O. O. 9 O.2 O.6 0.4 O.S. O. 12.8 1.8 Extra Slice #3 22 O.3 O.8 O. 6 - 0.2 O.3 0.04 O.4 O.04 10.5 - 1.4 Extra Slice #4 2.4 + 0.4 O.7 O. .2 + 0.1 O.6 0.4 O3 + O.O3 7.3 O.7 Extra Slice #5 26 - 0.6 O.7 O. .2 + 0.1 O.2 O.O2 O3 + O.04 9.7 2.3 Extra Slice #6 26 - O.S O.9 O. 3 - 0.1 O.2 OO1 O3 + O.OS 5.7 O.7 Pituitary 10.1 - 0.8 8.2 - 18 8.4 + 1.1 2.2 O.3 2.8 O.S 42.6 3.4 Optic Chiasm 5.1 O.7 7.4 1.7 8.0 O.7 1.7 - 0.3 1.9 O.9 25.654 Dorsal Dura 27.6 3.0 15.3 2.6 31.1 - 4.O 4.6 O.9 5.8 1.8 121.2 - 8.1 Ventral Dura 23.5 - 31 15.0 - 2.5 32.3 - 4.4 4.0 + 1.1 114 - 3.8 105.0 - 8.8 Spinal Dura 47.2 3.0 2.8 O.3 3.3 O.7 O.S. O.1 O.7 O.1 4.7 1.2 Upper Cervical Spinal 2.0 O.2 12 O.1 2.O. O.3 O.4 0.04 O.6 O.2 14.7 - 2.7 Cord Lower Cervical Spinal 2.6 O.3 O.6 O.1 O.6 0.1 O.1 OO1 O.3 - 0.1 1.O. O.1 Cord Thoracic Spinal Cord 1.6 O.2 O4 O.1 O.S. O.1 O.1 OO1 O.2 O.OS O.9 O.1 Lumbar Spinal Cord 2.1 O.3 O3 + O.04 O.4 - 0.1 O.1 OO1 O.2 O.O2 O.7 O.1 Circle of Willis & 18.1 2.8 11.7 - 2.5 14.8 11 9.3 2.5 5.8 11 65.7 8.3 Basilar Artery Carotid Artery 33.23.3 14.1 + 2.0 16.1 2.3 3.1 - 0.8 6.3 0.4 38.1 24 Renal artery (L) 111.2 - 10.1 4.4 + 1.0 114 - 3.3 O.6 0.1 3.7 1.5 8.6 24 Superficial Nodes (2) 25.3 2.9 4.8 + 0.4 10.4 2.2 1.5 + 0.7 2.4 + 0.1 7.6 O.9 Cervical Nodes (2) 626 - 9.2 5.6 O.7 6.9 O.8 1.1 O2 2.6 O.O3 25.1 3.7 Axillary Nodes (2) 42.8 12.8 3.7 O.S 6.0 - 0.6 O.S. O.1 2.6 O.6 4.3 O.7 Blood Sample 1,361 + 42.5 13.9 O.9 19.7 1.4 175 61 223 842 32.9 - 2.0 Muscle (R, deltoid) 19.1 - 3.8 2.7 - O.S 2.90.7 O.4 0.1 O.9 O.3 3.1 O.7 Liver (R, Superficial 135 - 23.7 1.7 O2 2.6 0.4 O.2 O.OS O.8 O.2 3.4 O.3 obe) Kidney (L, tip) 3SS 30.8 6.1 - 0.8 8.5 - 1.6 O.8 0.1 2.6 O.6 124 3.6 Orine 92.6 26.O 8.1 + 1.4 17.5 - 22 1.6 OS 6.3 1.7 124 2.5 Spleen (tip) 228 17.5 6.1 1.O 6.8 O.4 O.S. O.1 2.0 - O.S 8.7 2.O Heart 63.2 - 11.7 1.3 O2 2.7 - 0.6 O.3 0.04 O.6 O.1 2.5 O.8 Lung (R, top lobe) 261 - S13 2.9 - O.4 4.5 O.7 O.8 O.3 1.2 + 0.4 5.9 1.2 Thyroid 534 65.0 148 h 12.8 620 i 30.8 1,012 + 652 216 SO 285.8 43.2 US 9,556.260 B2 79 80 TABLE 8-continued Summary of tissue concentrations (nM E SE) of IN. IV, and Fab IgG at 30 min and 90 min endpoints with Outliers removed. Treatment IgG Protein (mean nM E SE IgG Microspheres (mean nM it SE). IgG FAB (mean nM t SE) Route Intravenous Intranasal Intranasal Intranasal

Time Point 30 min 90 min 30 min 90 min 30 min

Sample Size n = 7 n = 8 n = 6 n = 6 n = 5 n = 4

Esophagus 28.1 3.9 4.3 - 0.6 7.7 1.3 O6 O2 4.7 - 1.3 54 - O.7 Trachea 282 - 6.2 3.9 O.6 6.6 t 1.4 O6 O.1 2.0 - 0.6 7.6 1.5 Drug Standard CPM 7,448,243 + 7,630,853 + 7,166,204 + 6,799,540 + 6,861,351 + 7076679.3 128,562 169,309 76,377 303,198 210,321 47,472.8 Drug Standard CPM 7,089,796 + 7.470,182 + 7,200,437 + 6,872,480 + 6,758,588 + 709SO24.O. 272,234 171,868 154,753 300,896 176,717 69,509.2 Drug Standard CPM 7,390,784 + 7,689,073 + 7,022,761 + 7,059,120 + 7,027,097 + 71SO4S4.3 351,624 214,590 10,481 412,602 316,344 74,853.3

TABLE 9 Summary of tissue concentrations of IgG normalized to a 6 mg dose.

Treatment IgG FAB IgG Protein (mean nM IgG Microspheres (mean nM) (mean nM) Route Intravenous Intranasal Intranasal Intranasal

Time Point 30 min 90 min 30 min 90 min 30 min

Sample Size n = 7 n = 8 n = 6 n = 6 n = 5 n = 4 Volume Delivered (L) 47.7 47.4 47.6 48.0 48.0 48.2 uCi Delivered 69.5 69.6 7O.O 6O.O 59.7 76.4 mg Delivered 6.O 6.0 7.4 7.2 7.2 3.3 Olfactory Epithelium 43.0 441 288 272 2,660 569.O Respiratory Epithelium 41.1 136.213 132,671 62,371 103,758 1965O2.6 Anterior Trigeminal Nerve 1O.S 13.1 15.6 1.3 6.7 176.8 Posterior Trigeminal Nerve 6.3 6.0 6.8 O.9 2.6 54.7 Olfactory Bulbs 3.4 4.1 8.0 1.O 1.2 67.8 Anterior Olfactory Nucleus 1.9 1.5 2.1 O.S O.S 34.3 Frontal Cortex 2.9 1.4 3.1 0.4 O.S 32.3 Parietal Cortex 3.3 O.9 3 O.3 O.3 4.1 Temporal Cortex 2.9 1.1 .1 0.4 0.4 O.8 Occipital Cortex 2.3 1.8 2.0 O6 O.3 4.5 Extra Cortex 1.8 1.O .6 O.3 0.4 3.0 Amygdala 1.9 1.4 3 O2 O.3 21.2 Striatum 1.8 0.7 0.7 O.9 O.S O.2 Septal Nucleus 1.8 O.9 O.9 O.S O.S 4.2 Hypothalamus 2.0 1.7 .6 0.4 O.S 29.6 Thalamus 1.7 0.4 O.S O.1 O.3 1.8 Midbrain 1.8 0.7 .1 O2 0.4 S.6 Hippocampus 1.1 O.6 O.8 O2 0.4 O.6 Rons 1.7 O.9 3 O.3 0.4 8.5 Medulla 1.8 O.9 3 O2 O.3 7.1 Cerebellum 1.9 O.8 3 O6 0.4 3.2 Extra Slice #1 2.0 1.6 2.7 0.4 O.6 49.1 Extra Slice #2 2.1 1.O .6 O.S 0.4 23.2 Extra Slice #3 2.2 O.8 3 O2 O.3 9.1 Extra Slice #4 2.4 0.7 O.9 O.S O.2 3.3 Extra Slice #5 2.6 0.7 O O2 O.3 7.7 Extra Slice #6 2.6 O.9 O O2 O.3 0.4 Pituitary 10.1 8.2 6.8 1.9 2.3 77.5 Optic Chiasm S.1 7.4 6.5 1.4 1.6 46.5 Dorsal Dura 27.6 15.3 25.2 3.8 4.9 220.4 Ventral Dura 23.5 1S.O 26.2 3.3 9.5 190.8 Spinal Dura 47.2 2.8 2.7 0.4 O.6 8.5 Upper Cervical Spinal Cord 2.0 1.2 1.6 0.4 O.S 26.7 Lower Cervical Spinal Cord 2.6 O.6 O.S O.1 O.3 1.8 Thoracic Spinal Cord 1.6 0.4 0.4 O.1 O.2 1.7 Lumbar Spinal Cord 2.1 O.3 0.4 O.1 O.1 1.2 Circle of Willis & Basilar Artery 18.1 11.7 12.O 7.8 4.8 119.4 Carotid Artery 33.2 14.1 13.1 2.6 5.3 69.3 US 9,556.260 B2 81 82 TABLE 9-continued Summary of tissue concentrations of IgG normalized to a 6 mg dose.

Treatment IgG FAB IgG Protein (mean nM IgG Microspheres (mean nM) (mean nM) Route Intravenous Intranasal Intranasal Intranasal

Time Point 30 min 90 min 30 min 90 min 30 min

Sample Size n = 7 n = 8 n = 6 n = 6 n = 5 n = 4 Renal artery (L) 111.2 4.4 9.2 O.S 3.1 15.7 Superficial Nodes (2) 25.3 4.8 8.5 1.2 2.0 13.9 Cervical Nodes (2) 62.6 S.6 S.6 O.9 2.2 45.6 Axillary Nodes (2) 42.8 3.7 4.9 0.4 2.2 7.8 Blood Sample 1,361 13.9 16.0 146 186 59.8 Muscle (R, deltoid) 19.1 2.7 2.3 0.4 O.8 S.6 Liver (R, Superficial lobe) 135 1.7 2.1 O.2 0.7 6.1 Kidney (L, tip) 355 6.1 6.9 O6 2.1 22.6 Urine 92.6 8.1 14.2 1.3 5.2 22.6 Spleen (tip) 228 6.1 5.5 0.4 1.7 15.9 Heart 63.2 1.3 2.2 O.2 O.S 4.5 Lung (R, top lobe) 261 2.9 3.6 0.7 1.O 10.8 Thyroid 534 148 5O2 843 18O 519.6 Esophagus 28.1 4.3 6.2 O.S 3.9 9.9 Trachea 28.2 3.9 5.4 O.S 1.6 13.8

Results, Intranasal IgG Liquid Preparation Distribution at administration well and all survived until the 90 min desired 90 min End Point. Six rats received IN IgG liquid prepara- end point. The nanomolar IgG concentrations in tissues for tion at an average dose of 7.4 mg in 47.6 LL containing 70.0 IN IgG liquid preparation administrations taken at the 90 uCi with a 90 min end point. Animals tolerated the IN min end point are presented in Table 10. TABLE 10 Tissue concentrations (nM) of IgG after intranasal IgG liquid preparation administration at the 90 min end point with outliers excluded. BAX-24 BAX-33 BAX-34 BAX-3S BAX-36 BAX-40 Avg SE

Volume 47.8 47.4 47.4 47.5 47.5 47.8 47.6 O.1 Delivered (L) uCi Delivered 7O.O 70.O 7O.O 7O.O 70.O 7O.O 7O.O O.O1 mg Delivered 7.4 7.4 7.4 7.4 7.4 7.4 7.4 O.OO Olfactory 669.3 389.3 196.5 2O3.8 307.7 365.8 355.4 70.8 Epithelium Respiratory 205,721.0 194,945.7 189.621.1 139,524.3 150,482.2 101,469.9 163,627.4 +16,376.5 Epithelium Anterior 16.8 3O.O 1S.O 10.6 20.1 23.1 19.3 2.8 Trigeminal Nerve Posterior X 13.4 5.2 6.7 S.6 11.3 8.4 1.7 Trigeminal Nerve Olfactory 15.5 9.1 10.1 1O.S 3.6 10.8 9.9 +1.6 Bulbs Anterior 3.0 3.4 2.3 2.7 1.5 2.4 2.5 O.3 Olfactory Nucleus Frontal 3.3 5.9 2.4 S.O 2.7 3.3 3.8 O.6 Cortex Parietal 1.4 1.6 1.3 2.1 1.4 X 1.5 O.1 Cortex Temporal 1.1 O.9 1.7 1.4 1.2 2.0 1.4 O.2 Cortex Occipital 2.1 3.43 1.8 2.6 2.4 2.8 2.5 O.2 Cortex Extra Cortex 1.6 1.6 1.6 2.6 1.9 2.5 1.9 O.2 Amygdala 1.7 1.6 1.3 1.2 1.1 2.6 1.6 O.2 Striatum O.9 0.4 O.6 1.O 1.2 1.O O.9 O.1 Septal 1.4 1.1 1.1 O.9 1.4 O.9 1.1 O.1 Nucleus Hypothalamus 2.5 1.8 1.6 2.3 1.3 2.2 1.9 O.2 Thalamus O6 O.6 O.6 O.S O.6 O.78 O6 O.04 Midbrain 1.4 1.1 1.4 1.O 1.2 1.8 1.3 O.1 Hippocampus 1.O O.9 1.1 0.7 O.8 1.2 1.O O.1 US 9,556.260 B2 83 84 TABLE 10-continued Tissue concentrations (nM) of IgG after intranasal IgG liquid preparation administration at the 90 min end point with outliers excluded. BAX-24 BAX-33 BAX-34 BAX-3S BAX-36 BAX-40 Avg SE

Pons 1.9 1.2 1.1 1.9 1.2 2.2 1.6 O.2 Medulla 1.7 1.O 1.1 1.9 1.5 2.4 1.6 O.2 Cerebellum 1.4 1.O 1.6 2.1 1.4 2.5 1.7 O.2 Extra Slice #1 3.6 4.2 2.5 4.1 1.6 3.7 3.3 +0.4 Extra Slice #2 X 2.4 1.4 2.1 1.4 2.3 1.9 O.2 Extra Slice #3 1.4 1.9 1.1 1.6 1.1 2.3 1.6 O.2 Extra Slice #4 1.2 1.56 O.8 1.2 1.1 1.2 1.2 O.1 Extra Slice #5 1.2 1.3 O.9 1.1 1.1 1.43 1.2 O.1 Extra Slice #6 1.5 1.2 1.O 1.1 1.O 1.9 1.3 O.1 Pituitary 12.7 5.5 S.6 8.1 8.4 10.4 8.4 +1.1 Optic Chiasm 7.2 8.4 7.1 9.9 5.7 9.6 8.O O.7 Dorsal Dura 12.3 33.0 37.7 37.7 29.6 36.5 31.1 +4.0 Ventral Dura 21.6 47.6 41.2 26.5 21.4 35.4 32.3 +4.4 Spinal Dura 2.0 3.2 1.4 2.6 4.3 6.4 3.3 O.7 Upper 2.8 1.2 1.9 1.8 1.5 2.8 2.0 O.3 Cervica Spinal Cord LOWe 0.7 O6 0.4 0.7 0.7 X O.6 O.1 Cervica Spinal Cord Thoracic O.S 0.4 0.4 O.3 O.S O.9 O.S O.O8 Spinal Cord Lumbar 0.4 O.3 O.2 0.4 O.S O.8 0.4 O.O8 Spinal Cord Circle of 16.8 11.7 15.1 X 17.7 12.9 14.8 +1.1 Willis & Basilar Artery Carotid 17.4 13.5 12.6 13.0 13.5 26.9 16.1 2.3 Artery Renal artery 22.4 8.4 7.7 14.6 3.7 X 11.4 3.3 (L) Superficial 7.4 2O.S S.O 8.7 9.6 11.5 10.4 +2.2 Nodes (2) Cervical 6.7 8.9 4.4 5.2 6.9 9.1 6.9 O.8 Nodes (2) Axillary 7.2 5.8 4.3 4.5 7.9 6.3 6.O O.6 Nodes (2) Blood Sample 17.7 23.3 16.1 X 22.5 19.0 19.7 +1.4 Muscle (R, 2.6 3.2 1.O 1.3 5.4 3.6 2.9 O.7 deltoid) Liver (R, 2.8 1.4 3.9 1.1 2.7 3.4 2.6 +0.4 Superficial obe) Kidney (L, tip) 16.O 7.6 8.8 4.7 6.6 7.3 8.5 +1.6 Orine 25.9 10.8 18.7 12.3 18.0 19.3 17.5 +2.2 Spleen (tip) 5.8 7.8 6.8 7.1 5.3 7.7 6.8 +0.4 Heart 2.1 4.8 1.5 X 1.8 3.4 2.7 O.6 Lung (R, top 5.4 2.0 4.6 7.3 4.5 3.1 4.5 O.7 obe) Thyroid 543.6 566.7 700.5 X 680.7 606.1 619.5 30.8 Esophagus 13.5 8.7 6.3 7.4 5.5 4.8 7.7 1.3 Trachea 13.5 6.6 S.6 3.4 S.6 S.O 6.6 +1.4 Drug 7,390,846 7,130,719 7,130,719 6,977,049 6,977,049 7,390,846 7166204.3 +76,377.4 Standard CPM Drug 7,285,169 7,575,479 7,575.479 6,740,664 6,740,664 7,285,169 7200437.0 +154,753.0 Standard CPM Drug 6,990,473 7,032.426 7,032.426 7,045,383 7,045,383 6,990,473 7022760.5 +10,480.7 Standard CPM X = outlier removed from analysis

At the site of IN drug administration, the average IgG thalamus to a high of 1.9 nM in the hypothalamus. The concentrations in the respiratory and olfactory epithelia were 60 hippocampus was found to contain 1.0 nM IgG. A rostral to 163,627 nM and 355 nM respectively. A rostral to caudal caudal concentration gradient (3.3 nM to 1.2 nM) was gradient of 19.3 nM to 8.4 nM IgG was observed in the observed within the extra brain material sampled. Similarly, trigeminal nerve. A similar gradient from the olfactory bulb to the anterior olfactory nucleus of 9.9 nM to 2.5 nM IgG a rostral to caudal concentration gradient (2.0 nM to 0.4 nM) was observed. The average cortex concentration of IgG after 65 was observed in the spinal cord. The average concentration IN administration was 2.2 nM. Concentrations of IgG in of IgG in the dura of the brain was 31.7 nM compared to a other brain regions ranged from a low of 0.6 nM in the spinal cord dura concentration of 3.3 nM. Other tissues US 9,556.260 B2 85 86 sampled from the ventral skull, the pituitary and optic difficulty (lung, esophagus, and trachea) were consistent chiasm, contained 8.4 nM and 8.0 nM. IgG respectively. across animals. The blood concentration of IgG at the 30 min end point Results, Intranasal IgG Microsphere Preparation Distri was 19.7 nM. Concentrations of IgG in peripheral organs bution at 90 min End Point. Six rats received IN IgG ranged from a low of 2.6 nM in the liver to a high of 7.7 nM microsphere preparation at an average dose of 7.2 mg in 48.0 in the spleen, with urine containing 17.5 nM. Concentrations LA containing 59.7 uCi with a 90 min end point. Animals of IgG in the basilar and carotid arteries were similar to the tolerated the IN administration well and all survived until concentration in the renal artery (14.8 and 16.1 nM versus the 90 min desired end point. The nanomolar IgG concen 11.4 nM). Average concentration of IgG in the sampled 10 trations in tissues for IN IgG microsphere preparation lymph nodes was 7.8 nM. Levels of IgG in tissues measured administrations taken at the 90 min end point in five of the to assess variability of IN administration and breathing six rats are presented in Table 11. TABLE 11 Tissue concentrations (nM) of IgG after intranasal IgG microsphere preparation administration at the 90 min end point with outliers excluded. BAX-31 BAX-32 BAX-37 BAX-38 BAX-39 Avg SE Volume Delivered (IL) 48.0 48.0 48.0 48.0 48.0 48.0 OOO uCi Delivered 57.5 52.7 62.8 62.8 62.8 59.7 2.O mg Delivered 7.2 7.2 7.2 7.2 7.2 7.2 OOO Olfactory Epithelium 293.5 3,632.7 853.2 1898.5 9,281.1 3,191.8 +1,625 Respiratory Epithelium 169,083.6 169,807.3 128,471.3 69.460.0 85,723.8 124,509.2 +20,723 Anterior Trigeminal 11.1 5.9 10.7 7.6 4.5 8.0 1.3 Nerve Posterior Trigeminal 2.6 3.3 3.6 X 3.1 3.1 O2 Nerve Olfactory Bulbs 2.0 1.9 1.2 1.2 1.2 1.5 O2 Anterior Olfactory 0.7 O.S O.S 0.4 O.8 O6 O. Nucleus Frontal Cortex 0.7 O.8 0.4 O.8 O.6 0.7 O. Parietal Cortex O.3 X O.1 0.4 O.S O.3 O. Temporal Cortex 0.7 0.7 O.3 O.S O.S O.S O. Occipital Cortex O.6 0.4 O.1 0.4 O.2 O.3 O. Extra Cortex O.6 O.81 0.4 0.4 X 0.5 0. Amygdala O.2 X O.3 O49 O.S3 0.4 O. Striatum O.2 1.3 O.2 0.4 O.9 O6 O2 Septal Nucleus 0.4 1.9 O.1 O.2 X O6 +0.4 Hypothalamus O.6 O.8 0.4 0.4 O.9 O6 O. Thalamus O.2 O6 O.1 O.2 0.4 O.3 O. Midbrain O.3 O.S O.2 X O.8 O.S O. Hippocampus O.3 O.S O.2 O.2 1.O O.S O. Pons O.S 0.7 0.4 O.S O.S O.S O. Medulla O.S 0.4 O.3 0.4 O.S 0.4 O.04 Cerebellum O.S O.8 O.2 0.4 0.7 O.S O. Extra Slice #1 1.O 1.O 0.4 O.8 0.7 O.8 O. Extra Slice #2 0.4 OSO O.2 0.4 O.96 O.S O. Extra Slice #3 O.3 0.4 O.2 0.4 O.S 0.4 O.04 Extra Slice #4 O.3 X O.2 O.3 O.3 O.3 O.O3 Extra Slice #5 O.3 0.4 X O.S O.3 O.3 O.04 Extra Slice #6 0.4 X O.2 0.4 0.4 O.3 O.OS Pituitary 4.4 2.1 2.9 2.7 1.6 2.8 OS Optic Chiasm X 3.4 X 1.9 0.4 1.9 O.9 Dorsal Dura X 11.3 3.8 3.7 4.5 5.8 1.8 Ventral Dura 11.8 26.O 7.4 3.9 8.1 11.4 3.8 Spinal Dura O.6 O.8 0.7 X X 0.7 O1 Upper Cervical Spinal O.S O.3 1.1 O.9 O.3 O6 O2 Cord Lower Cervical Spinal O.2 O.2 O.1 0.4 O.6 O.3 O1 Cord Thoracic Spinal Cord O.1 O.1 O.1 O.3 O.3 O.2 O.OS Lumbar Spinal Cord X O.1 O.1 O.2 O.2 O.2 O.O2 Circle of Willis & Basilar 8.9 S.O 3.4 X 5.9 5.8 +1.1 Artery Carotid Artery 5.3 7.1 5.9 7.5 5.9 6.3 +0.4 Renal artery (L) 1.8 3.3 1.9 9.6 1.8 3.7 1.5 Superficial Nodes (2) 2.3 2.1 2.7 2.6 2.3 2.4 O1 Cervical Nodes (2) 2.5 2.6 2.7 2.5 2.7 2.6 O.O Axillary Nodes (2) 2.2 1.4 1.9 4.6 3.1 2.6 O6 Blood Sample 249.6 388.4 53.0 6.6 417.6 223.0 84.2 Muscle (R, deltoid) O.O O.9 1.2 X 1.5 O.9 O3 Liver (R, Superficial lobe) 1.1 O.S O6 O.S 1.5 O.8 O2 Kidney (L, tip) 1.6 1.9 1.3 3.7 4.3 2.6 O6 Orine 4.7 4.6 6.7 2.7 12.8 6.3 1.7 Spleen (tip) 1.4 1.5 O.8 2.9 3.4 2.0 OS Heart O.S 0.7 O.2 O.S O.9 O6 O1 Lung (R, top lobe) O.9 2.3 O.8 O.9 X 1.2 +0.4 US 9,556.260 B2 87 88 TABLE 11-continued Tissue concentrations (nM) of IgG after intranasal IgG microsphere preparation administration at the 90 min end point with outliers excluded. BAX-31 BAX-32 BAX-37 BAX-38 BAX-39 Avg SE Thyroid 1813 153.4 X 3.14.3 X 216.4 49.6 Esophagus 2.4 S.6 1.2 5.3 8.8 4.7 1.3 Trachea 1.7 1.6 O.9 3.7 X 2.0 O.6 Drug Standard CPM 6,696,942 6,103,589 7,168,742 7,168,742 7,168,742 6,861,351 +210,321 Drug Standard CPM 6,548.447 6,157,644 7,028,950 7,028,950 7,028,950 6,758,588 +176,717 Drug Standard CPM 6,631,733 5,962,084 7,513,889 7,513,889 7,513,889 7,027,097 +316,344 X = outlier removed from analysis

at the site of IN drug administration, the average IgG 15 Normalized to a 6 mg IN dose, Fab tissue concentrations concentrations in the respiratory and olfactory epithelia were were on average 19-fold higher in the brain than the liquid 124.509 nM and 3,191 nM respectively. A rostral to caudal IgG preparations. A Summary of the tissue concentrations of gradient of 8.0 nM to 3.1 nM. IgG was observed in the IgG normalized to a 6 mg dose is presented in Table 9. The trigeminal nerve. A similar gradient from the olfactory bulb three times smaller molecular weight of Fab versus intact to the anterior olfactory nucleus of 1.5 nM to 0.6 nM IgG IgG is likely responsible for the increased efficiency of direct was observed. The average cortex concentration of IgG after delivery from the nasal cavity to the CNS. If the Fab has IN administration was 0.5 nM. Concentrations of IgG in similar biological effects as IgG for the treatment of other brain regions ranged from a low of 0.3 nM in the Alzheimer's disease, it would be a promising candidate for thalamus to a high of 0.65 nM in the septal nucleus. The IN delivery. hippocampus was found to contain 0.5 nM IgG. The average 25 Comparisons of brain tissue concentrations (nM) after concentration of IgG in the extra brain material sampled was intranasal IgG liquid and microsphere preparations at 30 and 0.4 nM, similar to the average cortex concentration, and a 90 min end points are depicted in FIG. 2A and FIG. 2B. Results of IN and IV delivery of the liquid protein rostral to caudal concentration gradient was observed. Simi preparation after 30 min. On average, IN administration of larly, a rostral to caudal concentration gradient (0.6 nM to the IgG liquid preparation resulted in lower brain concen 0.2 nM) was observed in the spinal cord. The average 30 trations than an equivalent IV dose administered at the 30 concentration of IgG in the dura of the brain was 8.6 nM min end point (for example the average cortex concentration compared to a spinal cord dura concentration of 0.7 nM. of 1.3 nM vs. 2.6 nM). However, to achieve these brain Other tissues sampled from the ventral skull, the pituitary concentrations of IgG, IV administration resulted in blood and optic chiasm, contained 2.8 nM and 1.9 nM IgG concentrations that were a hundred times higher than IN respectively. 35 administration (1.361 nM. vs. 13.9 nM). Higher IgG con The blood concentration of IgG at the 30 min end point centrations in peripheral organs and systems were also was 223.0 nM. Concentrations of IgG in peripheral organs observed with IV vs. IN administration. For example, IgG ranged from a low of 0.6 nM in the heart to a high of 2.6 nM concentrations in the lymphatic system were ten times in the kidney, with urine containing 6.3 nM. Concentrations greater with IV vs. IN administration (43.6 nM vs. 4.7 nM). of IgG in the basilar and carotid arteries were similar to the 40 When normalizing tissue concentrations to blood, liver, or concentration in the renal artery (5.8 and 6.3 nM versus 3.7 lymphatic concentrations, it was apparent that IN adminis nM). Average concentration of IgG in the sampled lymph tration targets the central nervous system. The ratio of tissue nodes was 2.5 nM. Levels of IgG in tissues measured to concentrations to blood concentrations of intranasal and assess variability of IN administration and breathing diffi intravenous IgG is presented in Table 12. For example for culty (lung, esophagus, and trachea) were fairly consistent 45 frontal cortex, IN administration results in a 48 fold higher across animals. IgG levels in the thyroid varied greatly prior concentration than IV when normalizing for blood concen to the removal of outliers. tration, 40 fold higher when normalizing to liver concentra Overall, IN administration of the IgG liquid preparation tion, and 5 fold higher when normalizing to average lymph resulted in higher brain concentrations than the microsphere concentration. Intranasal administration increased IgG tar preparation when normalizing to a 6.0 mg dose with brain 50 geting about 50-fold more than IV administration (relative to concentrations ranging from 0.4 to 1.7 nM. A Summary of the blood) to areas of the brain known to accumulate the IN. IV and Fab data is presented in Table 8. This could B-amyloid and heme (both known to bind IgG) including the be explained by lower concentrations of the microsphere frontal cortex, hippocampus, and the blood vessel walls of IgG reaching the olfactory and respiratory epithelium. Intra the cerebrovasculature. Importantly, B-amyloid tightly binds nasal microsphere preparation also resulted in about ten 55 heme and heme is both a strong pro-oxidant and pro times higher concentrations of IgG in the blood than the inflammatory agent known to inactivate brain receptors liquid preparation. involved in memory. TABLE 12 Comparison of intranasal and intravenous targeting of IgG. Tissue to Blood Ratios Tissue to Liver Ratios Tissue to Avg. Lymph Ratios

IV IN INIV IV IN INIV IV IN INIV

Olfactory O.O32 31649 1OO2.1 O.319 266.6SO 836.2 O.986 94.481 95.8 Epithelium

US 9,556.260 B2 91 92 Eight rats received IV IgG liquid preparation at an aver dard error, and outliers because the blood concentration was age dose of 6.0 mg in 47.4 uL containing 69.5 LCi (diluted less than 20% of the value observed in all other animals, in saline to a total volume of 500 uL for injection) with a 30 Suggesting the IV infusion was not successful. Nanomolar min end point. Animals tolerated the IV administration well concentrations of intravenously administered IgG liquid and all survived until the 30 min desired end point. One preparation were measured in seven rats at the 30 min end animal (BAX-3) was removed from analysis of mean, stan point and presented in Table 13. TABLE 13 Tissue concentrations of intravenously administered IgG liquid preparation was measured in rats at the 30 min end point and Outliers were removed. BAX-5 BAX-7 BAX-9 BAX-10 BAX-11 BAX-13 BAX-15 Avg SE

Volume Delivered 47.0 47.0 48.0 48.0 48.0 48.0 48.0 47.7 O2 (IL) uCi Delivered 69.7 69.5 70.5 70.3 70.1 68.3 68.3 69.5 O3 mg Delivered 6.O 6.0 6.O 6.O 6.O 5.9 5.9 6.O O.O3 Olfactory 33.0 4O.S 40.4 43.0 56.5 32.O 55.5 43.O 3.7 Epithelium Respiratory 30.4 33.5 46.7 39.1 59.5 29.0 49.4 41.1 4.3 Epithelium Anterior 7.4 14.8 13.5 10.3 10.1 7.9 9.2 1O.S 1.O Trigeminal Nerve Posterior 4.2 11.O 8.3 6.7 5.4 3.5 4.8 6.3 1.O Trigeminal Nerve Olfactory Bulbs 2.2 2.8 5.5 3.7 3.2 9 4.2 3.4 OS Anterior Olfactory .1 2.1 3.3 8 1.9 .2 2.2 1.9 O3 Nucleus Frontal Cortex 2.5 4.0 3.2 8 2.7 .2 4.9 2.9 OS Parietal Cortex 3.3 5.2 3.0 .6 2.6 3 6.4 3.3 O.7 Temporal Cortex 7 3.7 2.5 2.2 5.8 .5 X 2.9 O.7 Occipital Cortex .9 2.8 2.5 2.3 X 8 X 2.3 O2 Extra Cortex .4 2.1 2.6 8 X .1 X 1.8 O3 Amygdala 5 1.9 X 2.1 2.0 8 2.2 1.9 O1 Striatum 2.4 1.6 1.6 3 1.5 8 2.6 1.8 O2 Septal Nucleus .6 1.4 2.0 6 X 2.0 2.2 1.8 O1 Hypothalamus .2 2.4 2.7 7 1.9 .5 2.7 2.0 O2 Thalamus 1.2 1.3 1.8 1.3 2.1 0.9 3.1 1.7 0.3 Midbrain .1 1.4 2.3 3 2.2 .1 2.9 1.8 O3 Hippocampus .1 1.3 O6 3 X .1 X 1.1 O1 Pons .1 1.6 2.4 .4 1.6 3 2.8 1.7 O2 Medulla .2 1.4 2.7 5 X .2 2.7 1.8 O3 Cerebellum 3 1.7 2.5 8 2.9 .2 X 1.9 O3 Extra Slice #1 5 2.8 2.7 7 2.2 3 2.1 2.0 O2 Extra Slice #2 .6 3.6 2.2 4 1.8 .2 2.7 2.1 O3 Extra Slice #3 .9 3.3 2.1 4 2.0 .1 3.6 2.2 O3 Extra Slice #4 .9 3.1 2.5 4 2.6 .1 4.3 2.4 +0.4 Extra Slice #5 7 3.0 2.1 5 3.4 .1 5.3 2.6 O6 Extra Slice #6 .9 2.4 2.2 .6 3.9 3 5.3 2.6 OS Pituitary 10.9 X 12.7 9.4 8.7 7.1 11.8 10.1 O.8 Optic Chiasm 5.9 5.2 8.4 3.9 4.7 2.9 4.6 S.1 O.7 Dorsal Dura 14.8 31.7 30.7 31.0 29.5 18.2 37.4 27.6 3.O Ventral Dura 16.4 31.0 34.4 19.6 18.9 13.8 30.4 23.5 3.1 Spinal Dura 52.3 45.9 54.8 37.6 X 53.4 39.5 47.2 3.O Upper Cervical 1.4 2.3 2.7 8 2.1 1.7 2.0 2.0 O2 Spinal Cord Lower Cervical 2.5 2.5 3.7 3.4 1.4 2.4 2.2 2.6 O3 Spinal Cord Thoracic Spinal 1.6 1.9 2.8 .1 1.O 1.2 1.3 1.6 O2 Cord Lumbar Spinal 2.8 1.8 2.5 2.0 1.3 1.3 3.1 2.1 O3 Cord Circle of Willis & 16.8 23.8 X 14.2 15.7 9.9 28.4 18.1 2.8 Basilar Artery Carotid Artery 21.7 33.8 37.2 37.5 37.4 43.9 20.6 33.2 3.3 Renal artery (L) 98.8 129.2 76.5 94.4 129.3 139.0 X 111.2 10.1 Superficial Nodes 20.3 29.6 22.9 31.9 35.3 12.6 24.1 25.3 2.9 (2) Cervical Nodes (2) 32.5 39.6 78.7 43.4 83.9 65.5 94.9 62.6 9.2 Axillary Nodes (2) 103.2 31.9 75.4 18.6 37.9 14.1 18.6 42.8 12.8 Blood Sample 1,224.9 1,234.2 1,543.3 1,322.6 1,364.7 1413.4 1422.9 1,360.9 42.5 Muscle (R, deltoid) 39.06 19.5 24.6 13.3 13.0 10.7 13.4 19.1 3.8 Liver (R, 742 72.2 11S.O 126.1 122.2 1863 247.5 1348 23.7 Superficial lobe) Kidney (L, tip) 347.7 313.9 287.3 4.59.1 397.7 441.0 238.9 355.1 3O8 Urine 32.9 1745 1873 41.1 68.3 122.8 21.1 92.6 26.0 Spleen (tip) 234.3 241.9 196.8 317.5 2326 175.8 198.1 2281 17.5 Heart 57.7 42.1 87.5 53.5 44.2 35.6 122.1 63.2 11.7 Lung (R, top lobe) 392.8 289.6 219.1 104.5 482.5 177.0 1614 261.0 51.3 Thyroid 317.8 651.8 832.2 S22.9 545.5 372.O 496.9 534.2 65.O US 9,556.260 B2 93 94 TABLE 13-continued Tissue concentrations of intravenously administered IgG liquid preparation was measured in rats at the 30 min end point and outliers were removed. BAX-5 BAX-7 BAX-9 BAX-10 BAX-11 BAX-13 BAX-15 Avg SE

Esophagus 24.8 41.3 28.0 42.8 24.4 2O.S 15.1 28.1 3.9 Trachea 14.6 29.2 17.9 39.2 13.4 59.0 24.2 28.2 6.2 Drug Standard 7,378,277 7,493,218 7,635,815 7,367,611 7,809,027 6,770,035 7,683,717 7,448,243 +128,561.8 CPM Drug Standard 7,962,330 7,709,707 6,369,627 6,846,596 6,401,005 7,249,509 7,089,796 +272,233.7 CPM Drug Standard 7,947,735 8,077,594 6,447,049 6,626.261 7,855.283 7,390,784 +351,624.3 CPM X = outlier removed from analysis 15 The blood concentration of IgG at the 30 min end point a pre-weighed conical tube (15 mL) and stored on ice. was 1,361 nM. Concentrations of IgG in the respiratory and Triplicate 25 uL samples were removed for gamma count olfactory epithelia were low as expected (43 nM and 41 nM ing. respectively). A rostral to caudal gradient of 10.5 nM to 6.3 2O The sample was centrifuged at 1,000xg (3,160 rpm) for nM. IgG was observed in the trigeminal nerve. A similar 10 minutes at 4°C. Blood supernatant was removed into a gradient from the olfactory bulb to the anterior olfactory pre-weighed ultracentrifuge tube and stored on ice. The nucleus of 3.4 nM to 1.9 nM IgG was observed. The average cortex concentration of IgG after IV administration was 2.6 extraction procedure was repeated on the blood pellet a nM. Concentrations of IgG in other brain regions ranged 25 second time (i.e. same Volume of homogenization buffer from a low of 1.1 nM in the hippocampus to a high of 2.0 added to conical test tube containing pellet, inverted several nM in the hypothalamus. The average concentration of IgG times to dislodge the pellet, transferred into glass homog in the extra brain material sampled was 2.3 nM, similar to enizer, homogenized with 15 passes, transferred to same the average cortex concentration, and a concentration gra dient was not observed. Similarly, a concentration gradient 30 pre-weighed conical test tube, centrifuged, and blood Super was not observed in the spinal cord and the average IgG natant removed). All blood Supernatant was pooled and concentration was 2.1 nM. The average concentration of IgG stored in the same pre-weighed conical tube. The extraction in the dura of the brain was 25.6 nM compared to a spinal procedure was repeated on the blood pellet a third time. cord dura concentration of 47.2 nM. Other tissues sampled 35 Triplicate 25 uL samples from pooled blood supernatant from the ventral skull, the pituitary and optic chiasm, contained 10.1 nM and 5.1 nM. IgG respectively. were remove for gamma counting. Concentrations of IgG in peripheral organs ranged from a 2 mL of the pooled blood supernatant was ultracentri low of 19.1 nM in the muscle to a high of 355.1 in the fuged at 5,000xg (7,071 rpm) for 90 minutes at 4°C. to in kidney, with urine containing 92.6 nM. IgG concentrations a 100 kDa filter tube. After the first two rats, it was found in basilar and carotid arteries were considerably lower than 40 that 2 mL took a lot of time to filter so for and animals that the renal artery (18.1 and 33.2 nM versus 111.2 nM). followed, we centrifuged only 1 mL of the pooled blood Average concentration of IgG in the sampled lymph nodes Supernatant. At the same time, 2 mL of the pooled blood was 43.6 nM. supernatant in the ultracentrifuged at 5,000xg (7,071 rpm) for 90 minutes at 4° C. to in a 30 kDa filter tube. After the Example 3 45 first two rats, it was found that 2 mL took a lot of time to filter so for and animals that followed, only 1 mL of the The Effect of IN and IV Delivery on the Intactness pooled blood Supernatant was centrifuged. of IgG And 2 mL of the pooled blood Supernatant was ultracen 50 trifuged at 5,000xg (7,071 rpm) for 90 minutes at 4° C. to A study was conducted to examine whether IgG remains in a 10 kDa filter tube. After the first two rats, it was found intact after IN and IV administration. Specifically, rats were that 2 mL took a lot of time to filter, for subsequent animals administered 'I radiolabeled IgG either intranasally or only 1 mL of the pooled blood Supernatant was centrifuged. intravenously and the total intact and degraded IgG was Triplicate 25 uL samples were removed for gamma counting determined 30 min after administration. 55 from the filtrate (100 kDa filter tube), the retentate (100 kDa Experimental Design: The rats were anesthetized and IgG filter tube), the filtrate (30 kDa filter tube), the retentate (30 was administered as described above in Example 2. Blood kDa filter tube), the filtrate (10 kDa filter tube) for gamma and brain was sampled and intact IgG was detected. counting, the retentate (10 kDa filter tube) for gamma Blood was sampled approximately 30 minutes after intra counting. nasal administration prior to perfusing with at least 100 mL 60 Each brain was removed (on ice), weighed, and placed of Saline containing protease inhibitors and serum was into a glass tissue homogenizer, the brain was manually processed. homogenized (40-50 passes) with homogenization buffer at Each blood sample (1.0 mL) was added to glass/tissue a 1:3 dilution (i.e., 2 mL buffer per g wet brain) and the homogenizer containing 2.0 mL of homogenization buffer homogenate was transferred into a pre-weighed conical tube (H.B., 10 mM tris buffer, pH 8.0 containing protease inhibi 65 (15 mL) and stored on ice. Triplicate 25 uL samples from tors) and aprotinin (100 uL per mL blood). The sample was brain homogenate were removed for gamma counting. The manually homogenized (30 passes) and then transferred into sample was centrifuged at 1,000xg (3,160 rpm) for 10 US 9,556.260 B2 95 96 minutes at 4° C. Brain Supernatant was removed into pre TABLE 1.4 weighed ultracentrifuge tube and stored on ice. The extraction procedure was repeated a second time on Summary of Intactness of IgG in the Brain and Blood. the pellet (i.e., added same volume of homogenization buffer IN IV to conical test tube containing pellet, inverted several times R1 R3 Avg R2 R4 Avg to dislodge the pellet, transferred into glass homogenizer, BLOOD homogenized with 20-30 passes, transferred to same pre % greater than 100 kD 30 36 33 123 113 118 weighed conical test tube, centrifuged, and removed Super 10 % greater than 30 kD 34 34 34 123 110 116 natant). All brain Supernatant was pooled and stored in the % greater than 10 kD 67 57 62 99 108 104 same pre-weighed conical tube. The extraction procedure BRAIN % greater than 100 kD 93 70 81 78 77 77 was repeated a third time on the pellets. Triplicate 25 L % greater than 30 kD 87 78 82 83 84 83 samples from pooled brain Supernatant were removed for 15 % greater than 10 kD 88 78 83 88 93 91 gamma counting. 2 mL of the pooled brain Supernatant was ultracentrifuged Example 4 at 5,000xg (7,071 rpm) for 90 minutes at 4° C. to in a 100 kDa filter tube. After the first two rats, it was found that 2 mL Effect of Intranasal Administration of IgG on took a lot of time to filter, for Subsequent animals only 1 mL Amyloid Plaque Loads of the pooled blood supernatant was centrifuged. At the same time, 2 mL of the pooled brain Supernatant in the A study was conducted to examine whether intranasal administration of IgG decreases amyloid plaque loads in the ultracentrifuged at 5,000xg (7,071 rpm) for 90 minutes at 4 25 brain in vivo. The purpose of the study was to determine C. to in a 30 kDa filter tube. After the first two rats, it was whether chronic treatment with intranasally delivered IgG at found that 2 mL took a lot of time to filter, for subsequent two doses (0.4 g/kg/2 wk and 0.8 g/kg/2 wk) would have any animals only 1 mL of the pooled blood Supernatant was effect on the amyloid plaque load in a transgenic amyloid mouse model of Alzheimer's disease. centrifuged. And 2 mL of the pooled brain Supernatant was 30 Experimental Design: The TG2576 (“TG”) amyloid ultracentrifuged at 5,000xg (7,071 rpm) for 90 minutes at 4 mouse model was used in this study as a mouse model for C. to in a 10 kDa filter tube. After the first two rats, it was Alzheimer's disease and C57 mice were used as controls. found that 2 mL took a lot of time to filter, for subsequent TG2576 mice (cat. #1349-RD1-M) were acquired from animals only 1 mL of the pooled blood Supernatant was 35 Taconic, Inc. in two batches of 50 spaced one month apart (Batch 1 and Batch 2). Animals were individually housed centrifuged. with free access to food and water, and were kept on a 12 Triplicate 25uL samples were removed for gamma count hour light cycle. For dosing with drug in a mg/kg dosing ing from the filtrate (100 kDa filter tube), the retentate (100 scheme, mice were divided into three size classes within kDa filter tube), the filtrate (30 kDa filter tube), the retentate 40 each treatment group, Small, medium, and large. Size groups (30 kDa filter tube), the filtrate (10 kDa filter tube) for were made to include /3 of animals in each size group. Mice gamma counting, the retentate (10 kDa filter tube) for were re-evaluated to make new size groups every two gamma counting. weeks. The mice were divided into five treatment groups of 20 mice as described in Table 15. Results: Two rats received IV IgG liquid preparation and 45 two rats received IN IgG liquid preparation at an average TABLE 1.5 dose of 52 uL containing 56 uCi (diluted in saline to a total volume of 500 uL for IV injection) with a 30 min end point. Treatmentgroups assigned for intranasal administration of IgG. Animals tolerated the administration well and all survived Mouse Strain Drug Administration Description until the 30 min desired end point. 50 Tg2576 IN IgG 0.4 g/kg/2 wk “TG-High' Tg2576 IN IgG 0.8 g/kg/2 wk TG-Low' In the brain, approximately 80% of gamma counts from Tg2576 IN Saline (control) “TG-Saline' 'I-labeled IgG after both IN and IV delivery were greater C57 IN IgG 0.8 g/kg/2 wk “WT-High' than 100 kD, suggesting intact protein. In the blood, 100% C57 IN Saline (control) WT-Saline gamma counts from 'I-labeled IgG after IV delivery were 55 greater than 100 kD, suggesting all was intact. With IN The mice were ordered and received in the animal facility delivery, only 33% of gamma counts from I-labeled IgG at 2 months of age and were singly housed and aged for 6 found in blood was greater than 100 kD. Suggesting that months. At 8 months of age, the mice were acclimated to 'I-labeled IgG may be broken down and enter the blood as handling for awake intranasal delivery over the course of 1 part of the clearance process from the nasal cavity, the brain 60 month. Mice were then intranasally treated with IgG or or both. This also provides additional evidence that IgG saline three times/week for 7 months. At 16 months of age, entering the CNS after IN administration does not travel behavioral testing occurred for 5 weeks while intranasal from the nasal cavity to the blood to the brain, but rather treatment continued. At ~17 months of age, 12 mice/group along direct pathways involving the olfactory and trigeminal were euthanized and brain tissue was collected for analysis. nerves. A Summary of the intactness of IgG in the brain and 65 IgG and saline for IN delivery was prepared Friday blood after intranasal or intravenous administration is pre afternoons from stocks sent by Baxter, and stored at ~4° C. sented in Table 14. for use the following week. Solutions were made to deliver US 9,556.260 B2 97 98 a dose of either 0.4 mg/kg/2 wk IgG or 0.8 mg/kg/2 wk IgG, TABLE 16-continued and were made to deliver a total of 24 uL. Drug was also made to cater to each of the three size classes within a Sample Schedule for acclimation to awake IN drug delivery. treatment group. Day # Day Action Mice were acclimated to handling for a period of two-four 5 3 W Hold and pet -2-3 min weeks before the onset of intranasal dosing. Acclimation to 4 Th Hold and pet -2-3 min handling was important, as it helped ensure a correct body 5 F Lightly pinch scruff position for maximum effectiveness of awake intranasal 6 M Lightly pinch scruff drug delivery. In addition, mice that have not been properly 7 Tu Scruff and lift 10 8 W Scruff and lift accustomed to this process can have a severe anxiety reac 9 Th intranasal Grip tion after dosing. Mice spent about 1-3 days on each of nine 10 F intranasal Grip steps before progressing to the next step, depending upon the 11 M intranasal (IN) Grip and Invert animals comfort to handling. The mouse's stress level was 12 Tu intranasal (IN) Grip and Invert used as a measure of progress. This means monitoring the 13 W N Grip, Invert, empty pipette tip 15 14 Th N Grip, Invert, empty pipette tip mouse's movements, the amount/frequency of urination, 15 F N Grip, Invert, deliver 1 round saline to each nare defecation, trembling, and biting. If a mouse had a high 16 M N Grip, Invert, deliver 1 round saline to each nare stress response, it remained on that step before progressing 17 Tu N Grip, Invert, deliver 2 rounds saline to each nare to the next until the response is reduced. A sample acclima 18 W N Grip, Invert, deliver 2 rounds saline to each nare tion schedule can be seen in Table 16. Acclimation of the mice progressed through the following once-a-day steps. For awake intranasal delivery of drug, the intranasal grip, The steps were not performed more than once per day in each mouse was restrained twice and held with their necks order to minimize the anxiety in the mice. parallel to the floor while a volume of 24 ul of liquid was First, the mouse was placed in the palm of the hand for a administered. Specifically, un-anesthetized mice were period of two to three minutes, no more than one foot above grabbed by the scruff of their necks and held gently, but the cage top, as animals frequently jumped during this 25 firmly, in an inverted position so that the mouse cannot move introductory step. If the mouse attempted to crawl out of the around. Each mouse was given four 6 ul nose drops (alter hand and up one's arm, the mouse was lifted by the base of nating nares) using a 20-ul pipettor. Intranasal drug delivery the tail and placed back in one's hand. Second, the mouse began when mice were 9 months of age. was placed in the palm of the hand for three minutes and petted gently. The mouse was petted directionally from head 30 At 16 months of age, mice were subjected to a five week to tail, while allowing the animal to move about freely. battery of behavioral tests to assess for memory, sensorimo Third, the mouse was placed in the palm of the hand for tor, and anxiolytic changes. These included Morris water three minutes while massaging behind the ears (lightly maze hidden and visual platform tests (reference memory, pinching together the skin on the back of the neck using the visual ability), radial arm water maze (working memory), thumb and pointer finger). Fourth, the mouse was held/lifted 35 passive avoidance task (memory), Barnes maze (memory), by the scruff of its neck for 30 seconds, letting the mouse rest open field test (exploratory behavior), elevated plus maze on the cage top for 30 seconds before repeating the hold (anxiety), and rotarod (motor skills). again. Fifth, the mouse was held using the intranasal grip, After behavior, 12 mice from each treatment group were without inverting the animal, for a period of 30 seconds and euthanized and their brains collected for biochemical analy then released back to the cage top. This was repeated a 40 ses. These analyses include immunohistochemistry (IHC) second time after a one-minute rest period. Sixth, the mouse for amyloid plaques, inflammatory markers, and soluble and was held with the intranasal grip while inverting the animal insoluble amyloid. So its ventral side was facing up towards the ceiling with the Prior to euthanasia via transcardial perfusion, mice were animal's neck is parallel to the floor. This position was held anesthetized with sodium pentobarbital (60 mg/kg diluted for 30 seconds and was then repeated a second time after a 45 1:4 with sterile saline). A first booster of half the full dose one-minute rest period. If the mouse freed itself from the was given followed by additional quarter-dose boosters, if grip, the mouse was put back on the cage top and re-gripped. necessary. The level of anesthesia and sensitivity to pain was If the mouse's stress level increased too much, the mouse monitored every five minutes throughout the procedure by was returned it to the cage. Seventh, the mouse was held testing reflexes including pinching the hind paw and tail. with the intranasal grip, inverted and a pipettor with an 50 empty tip was briefly placed over each nostril for 30 Mice were then euthanized with transcardial perfusion with seconds. This step was repeated after a one-minute rest 15 ml ice cold saline (no protease inhibitor needed) and period. Eighth, the mouse was held with the intranasal grip, blood was collected from the heart. Briefly, the arms of the inverted, and intranasally administered 6 Jul of saline into the mouse were taped down. The skin was cut to expose the 55 sternum. A hemostat was used to hold the sternum while left and right nare. Ninth, the mouse was held with the blunt dissection scissors were used to cut vertically on both intranasal grip, inverted, and intranasally administered 6 ul sides of the sternum making an incision with a V-shape to of saline into the left and right nare twice placing the animal expose the heart. Blood was collected from the heart prior to back on the cage top in between. perfusion and processed into serum. A Small hole in the left 60 Ventricle was made using a 24-gauge cannula. The cannula TABLE16 was inserted into the aorta and held in place. Extension Sample schedule for acclimation to awake IN drug delivery. tubing (filled with 5 mL of 0.9% NaCl) was attached to the cannula and the animal was manually perfused with 15 ml Day # Day Action saline. 1 M Hold for -2-3 min 65 Blood was spun down and serum divided into two ali 2 Tu Hold for -2-3 min quots. One aliquot was 50 uL and will be eventually pooled and sent for analyses of overall health of the treatment US 9,556.260 B2 99 100 group. The remaining serum was placed into its own tube The DAPI solution was poured into coplanjar containing the and Snap frozen for other analyses. slides. The slides were incubate for 20 min at RT. The slides The brain was collected and hemisected Sagitally in a were rinsed quickly in PBS, then 2x10 min in washing mouse brain matrix. The left half of the brain was dissected buffer, followed by a 10 min incubation in PBS. into olfactory bulbs, cortex/hippocampus mix, septum, mid 5 brain/diencephalon, brainstem (down to the V of the upper Immediately after staining, the slides were then dehy cervical spinal cord), and cerebellum. These tissues were drated, cleared, and mounted. Specifically, the slides were placed into microcentrifuge tube and Snap frozen in liquid incubated in 95% ethanol for 5 minutes, 100% ethanol for nitrogen. The right half was left in the matrix and sliced 3 two five minute increments, xylene for three five minute mm from the centerline. The inner portion towards the increments, and mounted with a coverslip in DPX without center of the brain was post-fixed in formalin (in a 15 ml 10 letting the specimen dry. The mounted slides were stored at conical tube filled with 10 ml formalin) and sliced for IHC room temperature. analyses. The outer portion was Snap frozen in liquid nitro Images of the fluorescently stained plaques were captured gen for eventual analysis for inflammation. with the AZ100 Multizoom Macroscope with the C1 si The medial 3 mm sagittal section of the right half of the Spectral Confocal attachment and an AZ Plan Apo 4x mouse brain was fixed by placing them each into 20 mL of 15 10% formalin. These samples were fixed for several hours at objective. Initial localization and focusing of the hippocam room temperature and then overnight at 4° C. on slow pus and cortex was conducted through epifluorescence moving rocker. The fixed Sagittal brain sections were placed imaging using filters for the DAPI stain. The scope was then medial side down into labeled pathology cassettes. The Switched to confocal imaging using the 637 nm laser for pathology department at Region’s Hospital conducted the acquisition of the IHF-labeled amyloid. Fine tuning within paraffin processing and embedding (dehydrate, infiltrate the Z-axis for optimal signal detection was confirmed with a with paraffin, mount into paraffin blocks). The paraffin 512x512 pixel resolution. Images were then captured at blocks were blinded by coding/relabeling. 1024x1024 with the Nikon EZ-C1 Software and the raw The paraffin blocks were sectioned at a thickness of 5um image files were saved in Nikon’s “...ids' file format. Cor using the Leica RM2235 microtome and collected on Super 25 responding tiff files of the 637 nm channel were generated frost Plus microscope slides (Cardinal Health, cati M6146 using Fiji (Image.J). The tiff files were then converted to 8-bit PLUS). Seven sections were collected per mouse, with at images (from 16-bit) and the contrast was enhanced by 0.5% least/approximately 100 um of tissue removed between through batch processing (Macro programming) in Fiji tissue section collections (labeled 1-6 from, medial to lat (Image.J). eral). To increase the quality of the sections to be stained, a 30 Plaques were quantitated in selected regions of interest in dissection microscope was used to identify and remove one of the seven sections. the hippocampus and cortex by determining the average Slides were deparaffinized and hydrated. Specifically, the number of plaques detected in each region and by determin slides were placed in a glass staining jar rack for easy ing the percent area covered by plaques within each region. transfer between staining dishes. The paraffin wax was 35 Image processing and analysis was conducted in Fiji. removed with Xylene washes (clearing) and then hydrated Plaques were defined within Fiji by using the particle with ethanol/water. Specifically, the slides were washed in analysis and the threshold function to select a minimum xylene three times for five minute intervals, washed in 100% pixel value that defined each identified particle as qualifying ethanol two times for five minute intervals, washed in 95% as a plaque. These values were determined by analyzing ethanol one time for five minutes, rinsed in running water for 40 multiple positive and negative controls and verifying which five minutes, and rinsed in PBS for five minutes. values correctly identified the plaques in these control slides. Heat induced epitope retrieval (HIER) was used to pre The region of interest within each image was chosen by a treat the slides prior to antibody staining A Tris/EDTA Buffer blinded researcher who was instructed to place the region of (pH 9) was used. The slides were immersed in a steamer interest in the position that would maximize the inclusion of proof dish containing the Target Retrieval Solution (Tris/ 45 plaques. The size (pixels) and number of plaques identified EDTA pH 9) pre-warmed to 70° C. The dish with slides was were copied into excel for data analysis. The plaques were then placed in the steamer and incubate for 30 minutes at 97 then characterized by their relative size. The plaque sizes C. The steamer was turned off and allowed to cool to at least reported in this study refer to the calculated radius of a 65° C. The container of slides was removed from the plaque assuming the particle conformed to the shape of a steamer and allowed to cool for another 10-15 minutes. The 50 perfect circle. The number of plaques and percent area slides were then removed from the container and rinsed in covered by plaques calculated from each region of interest PBS for 10 min in a coplan jar. was used as a single data point in comparing the treatment Non-specific binding sites were then blocked with normal groups. Two tailed t-tests were used to assess the signifi serum blocking solution (300 LL/slide) for 1 hour in a cance between groups. humidity chamber. Sections were incubated in a humid box 55 Prior to staining the complete set of collected tissue with primary antibody against amyloid (purified Anti-Beta sections, an initial verification of the staining and micros Amyloid, 17-24 (4G8) Monoclonal Antibody, from Covance copy analysis was conducted with relevant staining controls. (SIG-39220)) at a 1:200 dilution in primary antibody dilu These controls included, a positive control using sections tion buffer (0.01 M PBS pH 7.2) for 1 hour at room from one of the transgenic mice receiving saline, negative temperature. Sections were incubated in secondary antibody 60 controls in which either the primary or secondary antibody (Goat anti-mouse IgG, Alexa Fluor 647 (2 mg/ml) from incubation was omitted from the staining procedure and a Invitrogen (A21235)) dilution buffer (0.01 M PBS, pH 7.2) negative control using sections from one of the wild-type with a 1:200 secondary concentration for 1 hr at room mice receiving saline. Additional controls, including the temperature. titration of primary and secondary antibodies and the com Slides were then counterstained with DAPI. Diluted 300 65 parison of different epitope retrieval methods have been nM DAPI in PBS was used. 1 ul of 14.3 mM DAPI stock conducted previously in our lab using these antibodies and was diluted into 48 ml PEBS, vortexed, and mix thoroughly. the same experimental procedure. US 9,556.260 B2 101 102 Tissue Supernatants were analyzed using kits from Life ments of the soluble and insoluble amyloid beta peptides Technologies (formerly Invitrogen; Carlsbad, Calif.; part is Af340 and AB42 were taken in wild type and Tg2576 KHB3482 (AB40) and KHB3442 (AB42)). Generally, the (amyloid mouse model) pre- and post-intranasal IgG admin proper dilutions were first determined with three samples istration. The purpose of the study was to determine whether from either TG or WT mice, and then all samples were run chronic treatment with intranasally delivered IgG at two at that dilution. Samples were quantified using a polynomial doses (0.4 g/kg/2 wk and 0.8 g/kg/2 wk) would have any equation fit to a standard curve. Quantities of AB measured effect on the amyloid plaque load in a transgenic amyloid in the wells were then corrected for dilutions and total mouse model of Alzheimer's disease. protein (as determined by a BCA assay). Experimental Design: As described in Example 4, the Results: Immunohistochemical measurement of amyloid 10 TG2576 (“TG”) amyloid mouse model was used in this plaques in brain tissue slices demonstrated that there was a study as a mouse model for Alzheimer's disease and C57 significant drug effect. Both groups of TG mice administered mice were used as controls. The handling of the mice, IgG intranasally had significantly decreased plaque loads in preparation of drug, and administration of drug was con the cortex (FIGS. 3A, 3B, and 3E). ducted as described above in Example 4. Nasal administration of both the low dose and high dose 15 The mice were divided into five treatment groups of 20 of IgG significantly reduced the total percent area covered mice as described in Table 15. At approximately 17 months by plaques in the cortex of TG2576 mice (FIG. 3A). The of age and 12 months of treatment, 12 mice from each percent area covered by plaques decreased by 25.7% (low treatment group were euthanized and the concentration of dose; p=0.014) and 24.3%, (high dose; p=0.037), respec the AB40 and AB42 amyloid peptides in the brains of the TG tively. The change in the percent area covered by plaques and control mice were measured by ELISA to determine was slightly more pronounced at 27.1% for the low dose and whether amyloid plaque concentrations changes could be 26.0% for the high dose when the minimum threshold for detected. defining a plaque was increased from a radius of 25 um to Af340 and AB42 were measured by ELISA using Invitro 50 Lum (p values of 0.01 and 0.026, respectively). The gen ELISA kits. The ELISA kits were stored in refrigerator decrease in plaque load was also found to be significant 25 until they were ready to use. The kits were removed from when the minimum threshold was set at 100 um (p values of refrigerator and allowed to warm to room temperature 0.035 and 0.021, respectively). A change in the percent area before use. covered by plaques was not apparent when the Smaller Standards and samples were run in duplicate. The samples plaques (less than 50 um radius) were used exclusively in the and standards were run in a protease inhibitor cocktail with analysis. Thus, plaque reduction in the cortex appears to be 30 1 mM AEBSF (a serine protease inhibitor). AEBSF was more pronounced plaques larger than 50 um. important because serine proteases can rapidly degrade AB The number of plaques in the cortex of both low dose and peptides. The samples were kept on ice until they were ready high dose IgG treatment groups showed a trend toward a to be applied to the ELISA Plate. decrease in the numbers of plaques detected (FIG. 3B). This Sample matrix has a dramatic impact on AB recovery. To decrease reached significance in the low dose IgG treatment 35 ensure accurate quantitation, the standard curves were gen group when Small plaques (less than 50 um radius) were not erated in the same diluent as the samples. A standard included in the analysis. Specifically, treatment with intra reconstitution buffer was prepared by dissolving 2.31 grams nasally administered IgG provided a significant reduction in of sodium bicarbonate in 500 mL of deionized water and the plaque load when the data were analyzed by inclusion of pH was adjusted using 2 N sodium hydroxide until the pH plaques having a radius of from 50 um to 100 um, greater 40 was 9.0. than 100 um, and greater than 50 um. The decrease in plaque The standards for a quantitative standard curve were load reached significance for the high dose IgG treatment prepared. The Hu AB42 Standard was used. The Hu AB42 group when the radius of analyzed plaques was set at greater Standard was allowed to equilibrate to room temperature than 100 Lum. (RT) and then reconstituted to 100 ng/mL with Standard In contrast to the results seen in the brain cortex, IgG 45 Reconstitution Buffer (55 mM sodium bicarbonate, pH 9.0). treatments did not result in a significant change in either the The standard mixture was Swirled and mixed gently and percent area covered by plaques or the numbers of plaques allowed to sit for 10 minutes to ensure complete reconsti detected in the hippocampus (FIGS. 3C and 3D). Although tution. The standard was then briefly vortexed prior to intranasal administration of both low and high dose IgG preparing standard curve. Generation of the standard curve appeared to result in a slightly reduced plaque load in the 50 using the AB peptide standard was performed using the same hippocampus, the reduction was minimal and did not reach composition of buffers used for the diluted experimental significance in this region of the brain. samples. 0.1 mL of the reconstituted standard was added to Immunofluorescent staining of amyloid plaques in the a tube containing 0.9 mL of the Standard Diluent Buffer and hippocampus and cortex of aged TG mice is depicted in FIG. labeled as 10,000 pg/mL. Hu AB40. The standard was mixed 3E. As show, there is a decrease staining for amyloid plaques 55 and then 0.1 mL of the 10,000 pg/mL standard was added to in the hippocampus and cortex in mice that were treated with a tube containing 1.9 mL Standard Diluent Buffer and low and high IgG doses compared to TG mice treated with labeled as 500 pg/mL. Hu AB40. Mix. The standard was saline. mixed and then 0.15 mL of Standard Diluent Buffer was added to each of 6 tubes labeled 250, 125, 62.5, 31.25, Example 5 60 15.63, 7.81, and 0 pg/mL. Hu AB40 to make serial dilutions of the standard. Effect of Intranasally Administered IgG on Soluble The samples were then prepared for the plate. Specifi and Insoluble AB40 and AB42 cally, the samples were remove from the freezer, allowed to thaw, and diluted to the desired dilution using dilution buffer A study was conducted to assess the efficacy of chronic 65 provided with the kit mixed with a protease inhibitor tablet. intranasal (IN) administration of IgG at two doses in a The samples were kept on ice until loaded into the wells on transgenic amyloid mouse model. Specifically, measure the plate. US 9,556.260 B2 103 104 The plates were labeled as being either AB40 or AB42 The absorbance of each well was read at 450 nm having with a sharpie. 50 ul of standards and sample were added to blanked the plate reader within 30 minutes after adding the the pre-labeled wells. 50 uL of Hu AB40 or AB42 Detection Stop Solution. The concentrations were determined using Antibody solution provided with the kit was added to each the standard curve. well. The plate was covered and incubated for 3 hours at 5 room temperature with shaking Shortly before the 3 hours Results: The ELISA plates for both AB40 and AB42 expired, the Anti-Rabbit IgG HRP Working Solution was purchased from Invitrogen- yielded consistent standard prepared. To make this, 10 uI of Anti-Rabbit IgG HRP curves. The best dilutions of brain Supernatant for samples (100x) concentrated solution was diluted in 1 mL of HRP for soluble AB40 and AB42, and insoluble AB40 and AB42 Diluent for each 8-well strip used in the assay and labeled as to W 10x, undiluted, 10000x, and 2500x, respectively. Brain Anti-Rabbit IgG HRP Working Solution. concentrations of each protein were analyzed by first deter The solution was thoroughly decanted from wells and the mining the concentration of the sample in the well in the wells were washed 5 times with 300 uL of wash solution. ELISA plate based on the standard curve. These values were The plates were banged hard on lab bench to be sure it was then corrected for dilution of supernatant, dilution from the dry. 100 uL of the Anti-Rabbit IgG HRP working solution is extraction process, and then given a correction factor from was added to each well. The plate was covered and allowed a BCA analysis of total protein extracted. For each protein, to sit at room temp for 30 min. The solution was thoroughly between 1 and 4 samples were excluded for either being decanted from wells and the wells were washed 5 times with statistical outliers or being too high/low to fit within the 300 uL of wash solution. The plates were banged hard on lab standard curve. A summary of the soluble and insoluble bench to be sure it was dry. 100 uL of Stabilized Chromogen A?40 concentrations are presented in Table 17 and Table 18. was added to each well and the plate was immediately A summary of the soluble and insoluble AB42 concentra placed in the dark and allowed to sit for 20 min. 100 uL of tions are presented in Table 19 and Table 20. The ratios of Stop Solution was added to each well and the sides of the soluble AB40/A342 are provided in Table 21 and the ratios plate were gently tapped to mix. of insoluble AB40/A342 are provided in Table 22. TABLE 17

Soluble AB40 detected in brain.

Mouse sac Date Concentration Mouse sac Date Concentration order # Group measured (pg/ml) order # Group measured (pg/ml)

1 TG-Low 1-Oc 9078 33 TG-Saline 1-Oc 3940 6 TG-Low 1-Oc 3964 38 TG-Saline 1-Oc 1328 11 TG-Low 1-Oc 3110 43 TG-Saline 1-Oc 1983 16 TG-Low 1-Oc 2788 48 TG-Saline 1-Oc 3656 21 TG-Low 1-Oc 3934 53 TG-Saline 1-Oc 66SO 26 TG-Low 1-Oc 3747 58 TG-Saline 1-Oc 61.59 31 TG-Low 1-Oc 3796 4 WT-High 9-Oc O 36 TG-Low 1-Oc 5450 9 WT-High 9-Oc O 41 TG-Low 27-Sep 5261 14 WT-High 9-Oc O 46 TG-Low 1-Oc 2082 19 WT-High 9-Oc O 51 TG-Low 1-Oc 252O 24 WT-High 9-Oc O 56 TG-Low 1-Oc 9448 29 WT-High 9-Oc O 2 TG-High 1-Oc 3061 34 WT-High 9-Oc O 7 TG-High 1-Oc 1814 39 WT-High 9-Oc O 12 TG-High 1-Oc 4681 44 WT-High 9-Oc O 17 TG-High 1-Oc 2509 49 WT-High 9-Oc O 22 TG-High 1-Oc 7869 54 WT-High 9-Oc O 27 TG-High 1-Oc 6363 59 WT-High 9-Oc O 32 TG-High 1-Oc S541 5 WT-Saline 9-Oc O 37 TG-High 27-Sep S190 10 WT-Saline 9-Oc O 42 TG-High 1-Oc 3609 15 WT-Saline 9-Oc O 47 TG-High 1-Oc 1122 20 WT-Saline 9-Oc O 52 TG-High 1-Oc 12163 25 WT-Saline 9-Oc O 57 TG-High 1-Oc 1SO2 30 WT-Saline 9-Oc O 3 TG-Saline 27-Sep 3708 35 WT-Saline 9-Oc O 8 TG-Saline 1-Oc 4833 40 WT-Saline 9-Oc O 13 TG-Saline 1-Oc 1673 45 WT-Saline 9-Oc O 18 TG-Saline 1-Oc 4039 50 WT-Saline 9-Oc O 23 TG-Saline 1-Oc 2373 55 WT-Saline 9-Oc O 28 TG-Saline 1-Oc 41.33 60 WT-Saline 9-Oc O

Average Stod deviation Std error

TG-Low 4598.418 2395.218 6914399 TG-High 3932.644 1782.644 630.2598 TG-Saline 37O6.334 1570.737 473.595 WT-High O O O WT-Saline O O O

US 9,556.260 B2 109 110 TABLE 21-continued all TG mice, the concentration of insoluble AB40 and AB42 was much higher than soluble AB40 and AB42, roughly Ratios of soluble AB40/AB42. about 5000 and 7500 times higher, respectively. 42 TG-High O.243714 15 WT-Saline O Regarding group comparisons among the three TG 47 TG-High O.258574 20 WT-Saline O groups, there were no significant differences among any of 52 TG-High O.2SO769 25 WT-Saline O the groups for either soluble or insoluble AB40 or AB42 57 TG-High O.2S651 30 WT-Saline O using an ANOVA. This was somewhat surprising for 3 TG-Saline O.2132 35 WT-Saline O insoluble amyloid as there were clear differences in plaques 8 TG-Saline O.204815 40 WT-Saline O in the cortex between drug-treated and saline-treated TG 13 TG-Saline O.335658 45 WT-Saline O 10 mice. The most likely explanation is that the ELISA was not 18 TG-Saline O.181432 50 WT-Saline O as sensitive to these differences as the IHC slides of plaques. 23 TG-Saline O.219518 55 WT-Saline O 28 TG-Saline O.1783 60 WT-Saline O Example 6 Average Std deviation Std error 15 TG-Low O.198873 O.O7SOO8 O.O217 Effect of Intranasal Administration of IgG on TG-High O-20664 O.OS2O4 O.O157 Weight and Survival TG-Saline O.217088 O.061325 O.O177 WT-High O O O A study was conducted to assess the efficacy of chronic WT-Saline O O O intranasal (IN) administration of IgG at two doses in a transgenic amyloid mouse model. The purpose of the study was to determine whether chronic treatment with intrana TABLE 22 sally delivered IgG at two doses (0.4 g/kg/2 wk and 0.8 25 g/kg/2 wk) would have any effect on the mouse weight and Ratios of insoluble AB40/AB42. survival. Mouse Ratio of Mouse Ratio of Experimental Design: As described in Example 4, the S80 AB42. S80. AB42 TG2576 (“TG”) amyloid mouse model was used in this order # Group AB40 order # Group AB40 study as a mouse model for Alzheimer's disease and C57 1 TG-Low O438O6 33 TG-Saline O.14524 30 mice were used as controls. The handling of the mice, 6 TG-Low O.62584 38 TG-Saline O.31072 preparation of drug, and administration of drug was con 11 TG-LOW 0.34875 43 TG-Saline O.26396 ducted as described above in Example 4. 16 TG-Low O.19763 48 TG-Saline O.31298 21 TG-Low 0.37995 53 TG-Saline O.3551.6 The mice were divided into five treatment groups of 20 26 TG-Low O.13045 58 TG-Saline O.30663 mice as described in Table 15. The weight and survival of the 31 TG-Low O.366.82 4 WT-High O.41437 35 mice were monitored for 103 weeks. The weight of each 36 TG-Low O.21269 9 WT-High 1.10685 41 TG-Low O.22742 14 WT-High O.80589 mouse was recorded weekly (data not shown). 46 TG-Low O.2882 19 WT-High O. 62142 Results: These experiments showed that intranasal IgG 51 TG-Low O.25915 24 WT-High 0.30555 increases the lifespan of TG mice. FIG. 4A shows that TG 56 TG-Low O62688 29 WT-High O.62708 mice have an increased lifespan when they are administered 2 TG-High O44986 34 WT-High 0.66754 40 7 TG-High O.17399 39 WT-High 0.798.17 a high (0.8 g/kg/2 wk) or a low (0.4 g/kg/2 wk) dose of 12 TG-High O.5557 44 WT-High 0.5905 intranasal IgG compared to TG mice administered saline 17 TG-High O-36116 S4 WT-High 1.26484 intranasally (control). FIG. 4B shows that TG mice admin 22 TG-High O.21629 59 WT-High 1.00566 27 TG-High O.17166 5 WT-Saline 1.0335 istered intranasal IgG had longer lifespans than WT mice. 32 TG-High O.14659 10 WT-Saline Although this study begun with 20 mice in each cohort, due 37 TG-High O.29242 15 WT-Saline 0.88232 45 to the mass euthanasia performed to evaluate amyloid 42 TG-High 0.22767 2O WT-Saline 0.35914 plaque content (as described in Example 5), Kaplan-Meier 47 TG-High O.25291 25 WT-Saline 52 TG-High O.32982 30 WT-Saline 0.92744 Survival analysis was performed using the Sub-group of 8 57 TG-High O.151.9S 35 WT-Saline mice in each cohort that were not euthanized. Dosing to the 3 TG-Saline O.24128 40 WT-Saline 0.82407 mice in the Sub-groups was continued as described above 8 TG-Saline O.2226 45 WT-Saline 1.06434 50 through the entirety of the experiment. 13 TG-Saline O.32O33 50 WT-Saline 0.958.64 18 TG-Saline 0.3O277 55 WT-Saline 1.02571 28 TG-Saline O.15252 60 WT-Saline 1.10371 Example 7 Average Std deviation Std error 55 TG-Low O.3418.191 O.15905 O.O4591 Effect of Intranasal Administration of IgG on TG-High O.2727456 O.132S6 O.04.192 Memory TG-Saline O.2667446 O.O6933 O.O2OO1 WT-High 0.7461702 O.28.933 O.O8724 WT-Saline O.9087633 O.22479 O.O7493 A study was conducted to examine whether intranasal 60 administration of IgG affects the memory in the brain in vivo. The purpose of this study was to examine whether The most obvious and expected result was that both chronic treatment with intranasally delivered IgG at two soluble and insoluble AB40 and AB42 were drastically doses (0.4 g/kg/2 wk and 0.8 g/kg/2 wk) would have any higher in all TG mice than WT mice. Soluble AB40 and effect on memory in a transgenic amyloid mouse model of Af342 were not detectable in WT mice, while insoluble AB40 65 Alzheimer's disease. and AB42 were present, though at about 1000 times lower Experimental Design. At 15 months of age, the mice than in TG mice. The next most obvious result was that in described in Example 4 were subjected to a six week battery US 9,556.260 B2 111 112 of behavioral tests to assess for memory, sensorimotor, and 4 trials/day). Before trials began, the mice were acclimated anxiolytic changes. These included Morris water maze hid to swimming in the water. For each of these blocks of trials, den and visual platform tests (reference memory, visual mice were randomly dropped into four quadrants within the ability), radial arm water maze (working memory), passive MWM (round tub with water) and allowed to swim for 60 avoidance task (memory), Barnes maze (memory), open 5 seconds or until they reached the platform. The mouse's field test (exploratory behavior), elevated plus maze (anxi ability to reach the platform depended on his ability to ety), and rotarod (motor skills). remember visual cues from previous trials and their location Results: For each behavioral test, comparison data was in relation to the platform. Mice that did not reach the analyzed using T-tests as described above in Table 23. platform after 60 seconds were placed on the platform. Mice Statistical tests were performed on data after removal of 10 were allowed to remain/rest on the platform for 20 seconds both statistical outliers and non-compliant mice, which were between trials. All data was recorded using Mouse App specified for each behavioral test. Data was first analyzed by Software, which records escape latency. comparing WT-saline (WT-Sal) mice to TG-saline (TG-Sal) The Morris Water Maze Visual Platform is designed to mice to determine whether there is a transgenic (model) assess visual ability. It was run just like the MWM hidden effect for that test. Comparisons between all TG and all WT 15 platform, except the platform was raised just above the mice were also performed. Although the latter analysis is Surface of the water, has a flag on top to identify it, and confounded by drug treatment, it gains power by increasing stripes along the side to make it more visual. It was only run sample size and serves to give an overall picture of a for one day. Analysis was performed the same as with the potential transgenic (model) effect. Comparisons were made MWM hidden platform tests. among individual drug treatment groups. Specifically, the Overall, the Morris Water Maze Hidden Platform tests drug treated TG groups were compared directly to the showed that there was a clear trend of learning both through TG-Saline group to determine whether the drug had any out the week and during individual days, demonstrating that effect. the test was effective for measuring memory. Escape laten

TABLE 23

T-tests used to evaluate results of behavioral studies in wild type and Alzheimer's disease mouse models administered IgG intranasally.

Comparison Reason for Comparison

WT-saline vs. TG-Saline To determine whether there is a transgenic effect of the model.

WT-all vs. TG-all (all = saline and To provide a larger scale view of the transgenic effect of the IN IgG) model.

TG-saline vs TG-low dose IN IgG To determine whether TG mice treated with the low dose of performed differently than TG mice treated with saline.

TG-saline vs TG-high dose IN IgG To determine whether TG mice treated with the high dose of IgG performed differently than TG mice treated with saline.

Overall, in the three visio-spatial memory tests, mice cies were lowest during days 3 and 4, and were especially learned over time, and there was generally improved per 55 lowest during trials 3 and 4 on these days. formance in the WT mice as compared to the TG mice, which was expected. There was also a difference between There was evidence of a transgenic model effect. Table 25 WT and TG mice in the Elevated Plus Maze. There were and Table 26 show that both WT groups had lower escape minimal observed differences in the Rotarod and Open Field latencies than all three TG groups on days 3 and 4. WT-Sal Tests, but differences were not expected. Compliance was 60 mice had lower escape latencies than TG-Sal mice (Table 24, only a problem in the Barnes Maze, however, when non Table 25, Table 26, Table 27, and Table 28). However, when compliant mice were removed the learning trends were the WT and TG groups were put together, there were several present, and the model effect mirrored those seen in the significant differences, including B1-T2, B3–T4, B4-T1, MWM and RAWM. B4-T3, and B4-T4 (p<0.05 or 0.01; Table 24, Table 27, and The Morris Water Maze (MWM) Hidden Platform. 65 Table 28). Much of the power for this difference came from MWM is a standard test of spatial memory. MWM perfor the TG-high mice, which performed particularly well in this mance was assessed using hidden-platform testing (4 days, task. US 9,556.260 B2 113 114 TABLE 24

Summary of T-tests for specific comparisons in behavior tests. Tests are 2-sided and unpaired. Reported numbers are p-values. Gray cells p < 0.05; Boxed cells p < 0.1.

WT-Sal WT-Aws TG-Sal Measure Block Trial WSTG-Sal TG-All vsTG-High Escape 1 2 0.141 1 3 0.592 1

4 3 O.348

O.284 0.105 O.06 O.062 O.196 O.236 0.443 O.656 0.227 0.17 0.706 O.247 0.719 0.385 O.601 O.86 0.678 0.783

0.437 O.229 O.064 ().357 0.214 O.296 US 9,556.260 B2 115 116 TABLE 24-continued

Summary of T-tests for specific comparisons in behavior tests. Tests are 2-sided and unpaired. Reported numbers are p-values. Gray cells p < 0.05; Boxed cells p < 0.1.

MWM hid Escape 1 2 O.069 O.086 O.663 0.532

MWM 3 3 0.905 O.106 O.908 O.864 MWM 0.072 O.874 O.6 MWM O.102

MWM O.802 MWM O.192 Barnes | 1 || 1 || 0681 0.35 O.696

Barnes SC86 1 2 0.925 O.643 0.587 0.337 Barnes SC86 1 3 O.098 O.277 0.876 O408

Barnes O496 Barnes O.764 Barnes O.626 Barnes 0.657 Barnes O.606 Barnes O482 Barnes O.845 Barnes ().75 Barnes 0.416 Barnes O.603 Barnes 0.434 Barnes O.383 Barnes O.341 US 9,556.260 B2

TABLE 24-continued Summary of T-tests for specific comparisons in behavior tests. Tests are 2-sided and unpaired. Reported numbers are p-values. Gray cells p < 0.05; Boxed cells p < 0.1.

Barnes Errors 3 1 0.979 O.894 0.875 ().759 Barnes Errors 3 2 O.54 O.741 O.802 0.535 Barnes Errors 3 3 O.864 0.952 O.806 O.764 Barnes Errors 4 1 O.928 O.245 0.185 0.355

Barnes 4 0.885 0.965 0.736 0.758 Barnes O.116 O.19 ().707 MWM wis 1 1 O.074 0.589 MWM wis 1 2 ().507 ().597 MWM wis 1 3 O.863 0.959 MWM wis O.898 0.448 0.46 0.593 Open field Line na na O.534 O.138 O.112 O.688 Crossings Open field Velocity n/a na O.38 O.618 Elev. plus Time in 0.225 open arms Elev. plus Frequency O.13 in open 8S Rotarod Best run 0.875

Rotarod Average na na O.856 O.131 ().557 0.973 ill

Pass Avoid Escape 0.2O7

35 TABLE 25 TABLE 28 Average escape latencies (sec) from the Morris Water Maze tests. Average daily escape latencies (Sec) from the Morris Water Maze tests with noncompliance removed. Group Day 1 Day 2 Day 3 Day 4 40 Group Day 1 Day 2 Day 3 Day 4 TG-Low (N = 18) 34.54 30.47 25.03 24.68 TG-High (N = 18) 33.38 27.06 24.50 30.51 TG ALL (N = 45-49) 27.86 21.92 22.44 22.99 TG-Saline (N = 16) 24.80 22.2O 24.25 24.02 WT ALL (N = 28-30) 21.29 21.10 16.49 14.43 WT-High (N = 16) 23.38 23.58 17.73 16.11 WT-Saline (N = 18) 27.26 26.82 24.85 26.82 Like with RAWM, the three transgenic groups are 45 grouped closely in Table 25 and Table 26. The only signifi cant differences between TG-Sal and TG-low came on TABLE 26 B1-T1, B2-T2, and B4-T1 (Table 24), and in each case, Average escape latencies (sec) from the Morris Water Maze tests with TG-Sal mice had shorter escape latencies than TG-low mice, non-compliance removed. 50 who performed particularly poor in this task. There was only one example in which there was a statistical difference Group Day 1 Day 2 Day 3 Day 4 between TG-high and TG-Sal (B1-T1). In this instance, TG-Low (N = 15-18) 31.36 25.53 25.03 23:46 TG-Sal did very well and outperformed the TG-high mice. TG-High (N = 15-16) 28.73 20.55 2006 25.57 However, it should be noted that the WT-high mice consis TG-Saline (N = 14-15) 23.25 1968 21.87 1968 WT-High (N = 14-15) 20.93 19.30 14.92 13.18 55 tently outperformed all other groups in this task. Although WT-Saline (N = 13-16) 21.65 22.67 18.07 15.87 T-tests performed at each trial showed no statistical differ ences between WT-high and WT-Saline, repeated measures ANOVA would demonstrate a difference between these two groups. TABLE 27 60 For the MWM hidden platform test, the escape latency Averagedaily escape latencies (Sec) from the Morris Water Maze tests. (time to find the platform) was collected. T-tests were conducted for each day of each trial (1-4). Data was ana Group Day 1 Day 2 Day 3 Day 4 lyzed with non-compliant mice removed in order to more TG ALL (N = 52) 31.14 26.75 24.61 26.50 accurately represent memory. Non-compliant mice were WT ALL (N = 34) 25.43 25.29 21.50 21.78 65 defined as any mice that had escape latencies of 60 seconds (the full time allotted) for trials 3 and 4, when they should have been learning to some extent. The percent of non US 9,556.260 B2 119 120 compliant mice for each group was recorded. For hidden TABLE 31-continued platform tests non-compliance was as follows: TG-low-8.3%: TG-high-15.3%: TG-saline=7.8%; WT-high-7.8%; and WT-saline=18.1%. RAWM escape latency (seconds) of blocks 1 and 3. The Radial Arm Water Maze (RAWM). RAWM is used to ESCAPE LATENCY (BLOCK) evaluate short-term, working memory. Similar to a MWM, this test has a round tub with water, visual cues throughout T1(1) T3(1) T1(2) T3(2) T1(3) T3(3) T1(4) T3 (4) the room and a hidden platform. It is unique in that inserts are placed into the tank to create six radially distributed arms TG-High 49.72 44.94 47.78 42.11 40.OO 3S.S6 32.70 29.76 of equal size that emanate from the center. Before trials 10 (N = 18) began, the mice were acclimated to Swimming in the water. Mice were dropped into 4 radial arms, in an order selected TG-Saline SOS8 48.96 49.92 34.98 39.94 28.45 32.7S 28.04 randomly for each trial, and given 1 minute to find the platform, with 20 seconds of rest between each trial. Trials (N = 16) occurred daily for twelve days and each day the platform 15 WT-High 39.96 43.04 44.81 34.72 41.91 39.54 40.15 24.78 was moved to a new location. Halfway through the testing, an extra intra-maze visual cue was added to the tank in an (N = 18) effort to make the test a little easier. The visual cue was a WT-Saline 40.62 41.76 43.28 40.15 42.41 33.83 33.09 24.96 large X made of tape and placed on the inner wall of the (N = 18) maze above the arm with the escape platform. Both errors and escape latency were recorded. TABLE 29

RAWM escape latenc Seconds) of mice ouped in blocks 1-4.

Block 1 Block 2 Block 3 Block 4 T1(1) T2(1) T3(1) T4(1) T1(2) T2(2) T3(2) T4(2) T1(3) T2(3) T3(3) T4(3) T1(4) T2(4) T3(4) T4(4) TG-Low (N = 18) 49.2O SO.48 49.44 45.02 48.06 42.98 40.42 46.66 44.17 35.45 36.25 40.67 37.46 26.44 22.72 29.89 TG-High (N = 18) 49.72 47.13 44.94 47.33 47.78 34.09 42.11 42.96 40.OO 45.02 35.56 41.59 32.70 27.2O 29.76 29.57 TG-Saline (N = 16) 50.58 44.13 48.96 42.94 49.92 41.58 34.98 43.50 39.94 36.21, 28.45 32.98 32.7S 29.06 28.04 29.42 WT-High (N = 18) 39.96 47.11 43.04 42.02 44.81 41.61 34.72 41.70 41.91 33.52 39.54 34.93 40.15 28.28 24.78 24.43 WT-Saline (N = 18) 40.62 38.50 41.76 40.26 43.28 35.98 40.15 40.81 42.41 31.7O 33.83 33.7O 33.09 27.87 24.96 25.20

35 Overall, RAWM was too difficult for mice in blocks 1 and TABLE 32 2, as evidenced by a general trend for the escape latency not to go below about 35 seconds (Table 30). After the addition RAWM escape latency (seconds) of blocks 1 and 4. of the extra visual cue in blocks 3 and 4, a clear trend of ESCAPE LATENCY (BLOCK decreased time to find the platform and errors became 40 T1(1) T4(1) T1(2) T4(2) T1(3) T4(3) T1(4) T4(4) apparent in all treatment groups from trial 1 to trial 4 (Table TG-Low 49.2O 45.02 48.06 46.66 44.17 40.67 37.46 29.89 30, Table 31, and Table 32). This demonstrated that the test (N = 18) was effective for measuring memory. TG-High 49.72 47.33 47.78 42.96 40.OO 41.59 32.70 29.57 (N = 18) TG-Saline 50.58 42.94 49.92 43.50 39.94 32.98 32.75 29.42 TABLE 30 45 (N = 16) WT-High 39.96 42.02 44.81. 41.70 41.91 34.93 40.15 24.43 RAWM escape latency (seconds) of blocks 1 and 2. (N = 18) ESCAPE LATENCY (BLOCK WT-Saline 40.62 40.26 43.28 40.81 42.41 33.70 33.09 25.20 (N = 18) T1(1) T2(1) T1(2) T2(2) T1(3) T2(3) T1(4) T2(4) 50 TG-Low 49.2O SO.48 48.06 42.98. 44.17 35.45 37.46 26.44 (N = 18) There was clear evidence of a transgenic model effect in TG-High 49.72 47.13 47.78 34.09 40.OO 45.02 32.7O 27.20 RAWM (Table 33 and Table 34). In Table 35 an overall (N = 18) Summary of all groups averaged out overall days shows that TG-Saline 50.58 44.13 49.92 41.58 39.94 36.21 32.75 29.06 (N = 16) in all four trials, both WT groups had lower times to find the WT-High 39.96 47.11 44.81 41.61 41.91 33.52 40.15 28:28 55 platform than all three TG groups. This was also true of (N = 18) errors for trials 2-4 (Table 36). In Table 33, Table 34, Table WT-Saline 40.62 38.50 43.28 35.98 42.41 31.7O 33.09 27.87 35, and Table 36, individual blocks and trials can be seen. (N = 18) For escape latency, WT-Sal mice had significantly shorter escape latencies than TG-Sal mice in B1-T1 (Batch 1-Trial TABLE 31 60 1), B1-T3, and B3-T2 (p<0.05 or 0.1) (Table 24). For errors RAWM escape latency (seconds) of blocks 1 and 3. (Table 36), WT-Sal mice had significantly fewer errors than ESCAPE LATENCY (BLOCK TG-Sal mice in B1-T3, B3-T2, and B4-T2 (p<0.05 or 0.1) (Table 24). When all WT mice were combined and com T1(1) T3(1) T1(2) T3(2) T1(3) T3(3) T1(4) T3 (4) pared to all TG mice (irrespective of treatment), it was clear TG-Low 49.2O 49.44 48.06 40.42 44.17 36.25 37.46 22.72 65 that WT mice outperformed TG mice. When all days were (N = 18) combined, WT mice had shorter escape latency and fewer errors than TG mice in all trials (Table 35 and Table 36). US 9,556.260 B2 121 122 Similarly, in individual blocks and trials, all WT mice had (Table 24). When all WT mice were combined and com shorter escape latency and fewer errors in all trials in blocks pared to all TG mice (irrespective of treatment), it was clear 2-4 (Table 35 and Table 36). Statistically, WT mice had that WT mice outperformed TG mice. When all days were shorter escape latencies than TG mice in B1-T1, B3-T2, combined, WT mice had shorter escape latency and fewer B3-T4, and B4-T4 (p<0.05) (Table 24). Statistically, WT 5 errors than TG mice in all trials (Table 35). Similarly, in mice had fewer errors than TG mice in B1-T1, B3-T2, individual blocks and trials, all WT mice had shorter escape B3-T4, B4-T2, and B4-T4 (p<0.05) (Table 24). latency and fewer errors in all trials in blocks 2-4 (Table 35 and Table 36). Statistically, WT mice had shorter escape TABLE 33 latencies than TG mice in B1-T1, B3-T2, B3–T4, and B4-T4 10 RAWM escape latencies (seconds) recorded (p<0.05) (Table 24). Statistically, WT mice had fewer errors for 12 days of RAWM testing. than TG mice in B1-T1, B3-T2, B3–T4, B4-T2, and B4-T4 (p<0.05) (Table 24). TG ALL (N = 52 WT ALL (N = 36 The Barnes Maze. The Barnes maze is a visual memory Arm Arm Arm Arm Arm Arm Arm Arm 15 task based on finding an escape hole on a table, aided by 1 2 3 4 1 2 3 4 visual cues throughout the room. The table was round, Day 1 S1.67 S3.33 49.08 45.24 45.94 45.56 46.81 45.22 elevated 1 m from the floor, and had 40 escape holes spaced Day 2 51.1S 45.58 48.54 46.87 36.11 40.22 40.06 37.47 equally around the periphery of the table. One of these holes Day 3 46.60 43.19 45.60 43.41 38.86 42.64 40.33 40.72 had an escape box directly underneath, while the others were Day 4 SO.29 37.31 39.87 43.27 43.17 37.67 37.03 34.83 Day 5 49.85 40.62 38.27 44.76 41.94 38.14 36.61 44.08 20 open. The motivation to find the escape box was aversive Day 6 45.41 40.45 39.84 45.2O 47.OO 40.58 38.67 44.86 stimuli in the form of bright lights and fans blowing above Day 7 45.76 38.76 37.14 42.38 41.53 32.08 34.53 40.67 the surface of the table. The escape box was located in one Day 8 38.79 41.61 39.20 38.92 43.36 28.36 38.36 28.97 location for the duration of the study. The mouse was given Day 9 39.7S 36.79 24.71 34.63 41.57 37.39 37.17 33.31 4 days, with 3 trials/day to learn the location of the escape Day 10 34.42 29.90 29.69 29.94 39.81 27.19 27.SO 25.47 Day 11 34.13 23.69 24.10 31.15 35.5O 27.50 26.08. 28.94 25 box. Mice were given up to two minutes on the table to find Day 12 34.54 28.94 26.60 27.81 34.56 29.53 21.03 2003 the escape hole. If after 2 minutes they did not find the escape box, they were placed into the box. Both escape latency to find the hole and errors were recorded and TABLE 34 RAWM escape latencies (seconds) of blocks 1-4. Block 1 Block 2 Block 3 Block 4 T1(1) T2(1) T3(1) T4(1) T1(2) T2(2) T3(2) T4(2) T1(3) T2(3) T3(3) T4(3) T1(4) T2(4) T3(4) T4(4) TG ALL (N = 52) 49.81 47.37 47.73 45.18 48.54 39.45 39.32 44.40 41.42 39.04 33.62 38.62. 34.37 27.51. 26.79 29.63 WT ALL (N = 36) 40.29 42.81 42.40 41.14 44.06 38.80 37.44 41.26 42.16 32.61 36.69 34.31 36.62 28.07 24.87 24.81

TABLE 35 40 analyzed. Errors were defined as head-pokes through holes that do not have the escape box. 12 day average of RAWM eScape latencies (Seconds). Overall, the Barnes maze test did not work well for the Trial 1 Trial 2 Trial 3 Trial 4 mice in this study. This was the only behavior test in which TG ALL (N = 52) 43.53 38.35 36.89 39.46 45 non-compliance was an issue (roughly 50% of all mice did WT ALL (N = 36) 40.78 35.57 35.35 35.38 not perform the task). While running the tests, the mice were generally not scared of the aversive stimuli. However, among the mice that were compliant and included in the TABLE 36 analyses, there was a learning trend across the days and 50 trials, which can be seen in the escape latencies. 12 day average of RAWM errors (trial averages). There was evidence of a model effect with this test. Table Trial 1 Trial 2 Trial 3 Trial 4 37 and Table 38 shows that both WT groups have lower escape latencies on days 3 and 4 than all three TG groups. TG ALL (N = 52) 4.77 4.64 4.28 4.42 This mirrors data collected with the RAWM and MWM WT ALL (N = 36) 4.52 3.84 3.71 3.72 55 tests, the other two long-term memory tasks. This difference is also seen when all WT mice and TG mice were combined There was evidence of a TG model effect in RAWM. A as in Table 39 and Table 40. Summary of all groups averaged out over all days (Table 35) shows that in all four trials, both WT groups had lower times TABLE 37 to find the platform than all three TG groups. This was also 60 true of errors for trials 2-4 (Table 35 and Table 36). In Table Average escape latencies (see) from the Barnes Water Maze by treatment. 35 and Table 36, individual blocks and trials can be seen. For escape latency, WT-Sal mice had significantly shorter escape Time (s) Day 1 Day 2 Day 3 Day 4 latencies than TG-Sal mice in B1-T1 (Batch 1-Trial 1), TG-Low (N = 18) 105.15 99.76 95.44 85.67 B1-T3, and B3-T2 (p<0.05 or 0.1) (Table 24). As shown in 65 TG-High (N = 18) 107.74 94.57 100.30 97.33 Table 36, WT-Sal mice had significantly fewer errors than TG-Saline (N = 16) 95.48 89.10 90.10 82.31 TG-Sal mice in B1-T3, B3-T2, and B4-T2 (p<0.05 or 0.1) US 9,556.260 B2 123 124 TABLE 37-continued TABLE 41-continued Average escape latencies (Sec) from the Barnes Water Maze by treatment. Average number of errors from the Barnes Water Maze by treatment. Time (s) Day 1 Day 2 Day 3 Day 4 Day 1 Day 2 Day 3 Day 4 WT-High (N = 17) 99.06 95.98 93.65 82.04 TG-Saline (N = 16) 11.90 8.02 6.29 S.69 WT-Saline (N = 18) 94.15 97.41 93.43 87.63 WT-High (N = 17) 6.96 6.45 4.69 4.OO WT-Saline (N = 18) 946 8.44 5.35 4.89

TABLE 38 10 TABLE 42 Average escape latencies (sec) from the Barnes Water Maze by treatment with noncompliance removed. Average errors from the Barnes Water Maze by genotype. Time (s) Day 1 Day 2 Day 3 Day 4 Day 1 Day 2 Day 3 Day 4 15 TG-Low (N = 7-12) 82.81 78.25 83.60 72.75 TG ALL (N = 52) 8.97 6.31 5.53 S.O1 TG-High (N = 6-8) 84.28 72.14 75.71 68.86 WT ALL (N = 35) 8.25 7.48 S.O3 4.46 TG-Saline (N = 7-9) 79.71 65.38 74.30 71.48 WT-High (N = 8-10) 83.74 79.17 66.29 60.13 WT-Saline (N = 7-10) 69.17 68.43 73.00 60.74 For Barnes Maze, both the escape latency (time to find the escape hole) and errors (number of times a mouse pokes his head into a hole that does not have the escape box) were TABLE 39 collected. T-tests were conducted for each day of each trial (1-3). Data was analyzed with non-compliant mice removed Average escape latencies (sec) from the Barnes Water Maze by genotype. in order to more accurately represent memory. Non-compli 25 Time (s) Day 1 Day 2 Day 3 Day 4 ant mice were defined as any mice that had escape latencies of 120 seconds (the full time allotted) for trials 3, when they TG ALL (N = 52) 103.07 94.69 95.48 88.67 should have been learning to some extent. The percent of WT ALL (N = 35) 96.53 96.71 93.53 84.91 non-compliant mice for each group was recorded and was as follows: TG-low-48.6%; TG-high-61.1%; TG-sa 30 line=48.4%; WT-high-45.6%; and WT-saline=52.8%. TABLE 40 Elevated Plus Maze. The Elevated Plus Maze is a standard Average escape latencies (sec) from the Barnes Water test of baseline anxiety in which the animal is placed in the Maze by genotype with noncompliance removed. center of an elevated 4-arm maze that consists of two arms 35 that are open and two arms that are enclosed. The number of Time (s) Day 1 Day 2 Day 3 Day 4 times the animal entered each of the arms and the time spent TG All (N = 21-28) 82.OS 72.21 78.16 71.37 in each arm over 4 minutes was recorded. The test was used WT All (N = 17-19) 76.88 74.75 70.02 60.42 to determine the unconditioned response to a potentially dangerous environment (the open, unprotected arms) and There was no evidence of a drug effect in the Barnes Maze 40 anxiety-related behavior was measured by the degree to tests (Table 37, Table 38, Table 39, Table 40, Table 41, Table which the rodent avoids the open arms of the maze. 42). The only statistical significance was in B4-T1, in which There was a transgenic effect in the Elevated Plus Maze. TG-low mice performed very poorly and had longer escape In this model, all TG mice spent more time and made more latency than TG-Sal mice (p<0.1: Table 24). frequent arm entries into the open arms of the maze than all 45 WT mice, demonstrating inhibition of exploratory behavior TABLE 41 and anxiety that WT mice have regarding open spaces. When WT-Sal mice were compared to TG-Sal mice, TG Average number of errors from the Barnes Water Maze by treatment. mice spent significantly more time and have significantly Day 1 Day 2 Day 3 Day 4 50 more arm entries into the open arms (Table 24, Table 43, and TG-Low (N = 18) 8.48 5.57 6.24 5.39 Table 44). When all WT-mice and all TG-mice were com TG-High (N = 18) 6.85 S.S4 4.15 4.04 bined, the same results were seen (Table 44 and Table 45), p<0.05: Table 24). TABLE 43 Average time spent in open arms during the Elevated Plus Maze.

TIME (SEC

SUM PERCENTAGE Avg. Time Avg Time Std Error Std Error Avg. Time Avg Time Std Error Std Error Enclosed Open Enclosed Open Enclosed Open Enclosed Open TG-Low (N = 18) 115.2 31.4 10.8 5.3 48.0 13.1 4.5 2.2 TG-High (N = 16) 128.7 48.9 11.2 8.7 53.7 20.4 4.7 3.6 TG-Saline (N = 15) 117.5 34.6 11.6 7.4 49.0 14.4 4.9 3.1 WT-High (N = 16) 151.9 20.6 8.6 3.7 63.4 8.6 3.6 1.6 US 9,556.260 B2 125 126 TABLE 43-continued Average time Spent in Open arms during the Elevated Plus Maze.

TIME (SEC

SUM PERCENTAGE Avg. Time Avg Time Std Error Std Error Avg. Time Avg Time Std Error Std Error Enclosed Open Enclosed Open Enclosed Open Enclosed Open WT-Saline (N = 16) 1698 15.9 11.6 4.4 70.8 6.6 4.8 1.8 TG ALL (N = 49) 120.3 38.1 6.4 4.2 SO.2 15.9 2.7 1.8 WT ALL (N = 32) 1608 18.3 7.3 2.9 67.1 7.6 3.0 1.2

TABLE 44 Average frequency of entries into open arms during the Elevated Plus Maze. FREQUENCY SUM PERCENTAGE Avg. Freq Avg. Freq Std Error Std Error Avg. Freq Avg. Freq Std Error Std Error Enclosed Open Enclosed Open Enclosed Open Enclosed Open TG-Low (N = 18) 16.6 10.8 2.O 2.0 60.9 39.1 S.1 S.1 TG-High (N = 16) 16.O 14.8 2.2 3.6 57.8 42.2 5.2 5.2 TG-Saline (N = 15) 15.9 8.1 2.4 2.2 69.6 3O4 5.8 5.8 WT-High (N = 16) 13.8 3.4 1.5 O.S 82.1 17.9 2.4 2.4 WT-Saline (N = 16) 9.8 3.0 1.1 O.8 81.9 18.1 3.8 3.8 TG ALL (N = 49) 16.2 11.3 1.2 1.6 62.5 37.5 3.1 3.1 WT ALL (N = 32) 11.8 3.2 1.O O.S 82.O 18.0 2.2 2.2

There was no evidence of a drug effect in the Elevated TABLE 45-continued Plus Maze tests. Although the TG-high group had the most arm-entries and spent the most time in the open arms, it was Average velocity of mice. not significantly different from any other groups (Table 24, 35 Table 43, and Table 44). For the Elevated Plus Maze, both the time spent in open Avg. Velocity Std Dev Std Error and enclosed arms and the number of arm entries (also called frequency of arm entries) were recorded. Mice were not TG Saline (N = 15) 8.73 2.78 0.72 included in the analyses if they fell off the maze in less than 40 120 seconds. There were 3 mice that fell off, all from WT High (N = 16) 10.03 2.50 O.63 different groups. For outliers, mice were removed if both WT Saline (N = 17) 9.71 3.38 O.82 their time spent in open arms and frequency of entries into open arms were more than two standard deviations from the mean of their treatment group. Outliers included 3 mice, all from different groups. 45 TABLE 46 The Open Field Maze Test. The Open Field Maze Test is used to detect any change in spontaneous locomotor activity Average velocity of mice, averaged by genotype. due to drug treatment or anxiety. Each mouse was given 4 minutes to individually explore a rectangular box, while Avg. Velocity Std Error being tracked by the EthoVision video tracking system. For 50 analysis, the box was subdivided into 16 equally sized TG ALL (N = 51) 8.66 0.44 squares that are separated by manually drawn lines using the WT ALL (N = 33) 9.46 O.S6 “line draw” feature in EthoVision. The number of line crossings and patterns of exploration were measured. There was no evidence of a transgenic or drug effect in the 55 Open Field Maze tests. All groups of mice had very similar TABLE 47 line crossings and velocity (Table 24, Table 45, Table 46, Table 47, and Table 48). Average number of line Crossings by mice. 60 Avg Line TABLE 45 Crossings Std Dev Std Error Average Velocity of mice. TG Low (N = 18) 87.56 32.93 7.76 TG High (N = 18) 110.94 44.01 10.37 Avg. Velocity Std Dev Std Error TG Saline (N = 15) 105.53 29.47 7.61 WT High (N = 16) 113.06 30.10 7.53 TG Low (N = 18) 7.66 2.48 O.S8 65 WT Saline (N = 17) 112.59 33.44 8.11 TG High (N = 18) 9.32 3.73 O.88 US 9,556.260 B2 127 128 TABLE 48 TABLE 51-continued Average number of line Crossings by mice, averaged by genotype. Trial averages of run time (Sec) on the rotarod by treatment group. Avg Line Crossings Std Error Trial 1 Trial 2 Trial 3 TG ALL (N = 51) 102.65 5.33 TG ALL (N = 52) 15.72 20.44 33.72 WT ALL (N = 33) 107.83 6.21 WT ALL (N = 34) 16.60 25.63 24.60

For the Open Field Maze, both the number of line 10 There was no evidence of a drug effect among transgenic crossings and the overall velocity were measured. Outliers groups (Table 49, Table 50, and Table 51). However, it was were removed if an individual mouse's line crossings were observed that the WT-high mice had longer times on the more than 2 standard deviations from the mean of the rotarod than the WT-Sal mice. A t-test between WT-Sal and treatment group. This included 3 mice, each from different treatment groups. Analysis was performed for both line WT-high yielded a p-value of 0.089 for the longest run, and crossings and Velocity. 15 a p-value of 0.041 for the average run (T-tests not shown, The Rotarod Performance Test. The Rotarod Performance Table 49, Table 50, and Table 51). Testis used to detect any changes in endurance, balance, and For the Rotarod test, the time on the rotating bar before coordination. Mice were placed on an automated rotating the mouse fell off was recorded. Three trials were conducted. bar and allowed to walk on the bar for up to 60 seconds. The If a mouse reached 120 seconds (the maximum time) before speed of rotation was gradually increased and the rodents trial 3, Subsequent runs were not conducted. For each ability to remain on the rotating bar was recorded as the total treatment group, both the average time on the bar and the time spent on the bar. Mice were given three trials, and the maximum time on the bar for each mouse were analyzed. best time is used for analysis. Data could not be recorded if the mouse did not stay on the There was no transgenic model effect on the Rotarod tests. 25 rod long enough before starting (~3 seconds), and there was All groups performed essentially the same and there were no only 1 mouse that did not stay on long enough to start for all statistical differences among groups (Table 24 and Table 49, three trials. Table 50, and Table 51). There was a non-significant trend The Passive Avoidance Task. The Passive Avoidance Task for all WT mice to outperform all TG mice (Table 49, Table is a classical conditioning test used to assess short-term or 50, and Table 51). 30 long-term memory for mice and rats. The passive avoidance apparatus consists of equal-sized light and dark compart TABLE 49 ments with a light bulb fixed in the center of the roof of the light compartment. The floor consists of a metal grid con Longest average runs on the rotarod by treatment group. nected to a shocker. The two compartments are separated by Best Trial (Average) (sec) 35 a trap door. On the learning day (day 1), a mouse was placed TG-Low (N = 18) 30.59 in the light compartment and the time taken to enter the dark TG-High (N = 18) 37.33 compartment was recorded and termed as initial latency. TG-Saline (N = 16) 35.38 WT-High (N = 16) S4.13 Immediately after the mouse entered the dark chamber a WT-Saline (N = 18) 35.67 40 door was automatically closed and an electric footshock (0.7 TG ALL (N = 52) 37.33 mA) was delivered for 3 seconds. Twenty-four hours after WT ALL (N = 34) 35.38 the acquisition trial, a second retention trial was conducted and the time the mouse takes to enter the dark compartment as designated retention latency (RL, recorded to a maximum TABLE 50 45 of 500 seconds, no shock is administered during this entry). T-tests were performed to compare the effects of IN IgG WT Average run time on the rotarod by treatment group. VS. T.G. Avg. Time (Sec) Whereas RAWM, MWM hidden platform, and Barnes maze tests all showed evidence of learning and improved TG-Low (N = 18) 1943 50 TG-High (N = 18) 23.30 learning in WT mice over TG mice, this test consistently TG-Saline (N = 16) 22.35 showed the opposite effect, regardless of drug treatment. WT-High (N = 16) 35.24 WT-Saline (N = 18) 22.25 There was no evidence of a drug effect among transgenic TG ALL (N = 52) 23.30 groups (Table 24, Table 52, Table 53, and Table 54). WT ALL (N = 34) 22.35 55 TABLE 52 Passive avoidance learn day escape latency (Sec). TABLE 51 Learn Esc. St. Err Trial averages of run time (Sec) on the rotarod by treatment group. 60 TG-Low (N = 17) 445 9.4 Trial 1 Trial 2 Trial 3 TG-High (N = 19) 46.3 7.6 TG-Saline (N = 15) 43.7 9.2 TG-Low (N = 18) 1O.S3 21.25 27.06 WT-High (N = 17) 21.6 6.4 TG-High (N = 18) 15.72 20.44 33.72 WT-Saline (N = 18) 22.4 3.9 TG-Saline (N = 16) 16.60 25.63 24.60 TG ALL (N = 51) 44.9 4.9 WT-High (N = 16) 19.56 43.00 44.2O 65 WT ALL (N = 35) 22 3.6 WT-Saline (N = 18) 17.00 17.06 32.39 US 9,556.260 B2 129 130 TABLE 53 TABLE 55 Passive avoidance test day escape latency (Sec). Visual escape (Sec) by treatment group. Learn Esc. St. Err Group Trial 1 Trial 2 Trial 3 Trial 4 Average TG-Low (N = 15) 224.6 8.5 TG-Low (N = 18) 3483 3344 39.17 30.22 3344 TG-High (N = 17) 229.5 8.3 TG-High (N = 18) 31.44 33.67 35.33 37.89 33.67 TG-Saline (N = 13) 2O7.0 16.8 TG-Saline (N = 16) 23.19 36.56 29.75 28.94 36.56 WT-High (N = 16) 114.3 22.O WT-High (N = 16) 28.25 23:44 23.13 22.OO 23.44 WT-Saline (N = 18) 153.8 20.9 WT-Saline (N = 18) 29.78 29.06 26.11 25.50 29.06 TG ALL (N = 45) 2214 4.9 10 WT ALL (N = 34) 135.2 3.6 TABLE 56 TABLE 54 Visual escape (Sec) by treatment group, with non-compliance removed. 15 Passive avoidance average of escape latency differences (see). Group Trial 1 Trial 2 Trial 3 Trial 4 Average Average of TG-Low (N = 13) 30.46 29.15 31.15 18.77 29.15 Differences St. Err TG-High (N = 13) 21.69 27.92 25.85 29.38 27.92 TG-Saline (N = 14) 17.93 33.21 25.43 24.50 33.21 TG-Low (N = 15) 1902 8.5 WT-High (N = 14) 25.07 18.57 17.86 16.57 18.57 TG-High (N = 17) 1919 8.2 WT-Saline (N = 17). 31.41 27.24 24.12 23:47 27.24 TG-Saline (N = 13) 175.1 16.8 WT-High (N = 16) 98.8 22.4 WT-Saline (N = 18) 131.4 21.4 TG ALL (N = 45) 186.5 6.3 TABLE 57 WT ALL (N = 34) 116.1 15.5 25 Visual escape (Sec) by genotype group. This test demonstrated an unexpected TG effect. Whereas Group Trial 1 Trial 2 Trial 3 Trial 4 Average TG mice with impaired memory should normally have TG ALL (N = 52) 3O.O8 34.48 34.94 32.48 34.48 trouble remembering not to enter the dark chamber and WT ALL (N = 34) 29.06 26.41 24.71 23.85 26.41 receive a shock after training, this was not the case. TG mice 30 generally did not enter the chamber on the test day, whereas WT mice seemed not to care whether they received a shock TABLE 58 on the test day. These results can be seen in Table 52, Table 53, and Table 54. The poor performance of the WT mice Visual escape (Sec) by genotype, with non-compliance removed. compared to the TG mice is statistically significant (p<0.05; 35 Table 24). The same willingness for WT mice to enter the Group Trial 1 Trial 2 Trial 3 Trial 4 Average dark chamber can be seen in the learning phase and may play TG ALL (N = 40) 23.23 30.18 27.43 24.23 30.18 a role in the willingness of normal, WT mice to go receive WT ALL (N = 31) 28. SS 23.32 21.29 20.35 23.32 a painful shock. 40 For the Passive Avoidance Task, the escape latency on For the visual platform MWM, the escape latency (time to both the learning day (day 1) and the test day (day 2) were find the platform) was collected. T-tests were conducted for recorded and the difference between the escape latency each day of each trial (1-4). Data was analyzed with non between the test and learn day were calculated. Mice were compliant mice removed in order to more accurately repre not run on the test day (day 2) if they did not receive a shock 45 sent memory. Non-compliant mice were defined as any mice on day 1, which included 7 mice spread across 4 groups. that had escape latencies of 60 seconds (the full time allotted) for trials 3 and 4, when they should have been Mice did not receive a shock simply because they did not learning to Some extent. The percent of non-compliant mice enter the dark chamber. There were no outliers calculated. for each group was recorded. For visual platform tests Analyses were performed for the learn trial and the test trial. 50 non-compliance was as follows: TG-low-6.9%; Morris Water Maze Visual Platform. Differences in per TG-high-6.9%; TG-saline=3.1%; WT-high-3.1%; and WT formance in this test were not expected as all mice were saline=1.4%. genetically tested for the RD1 gene and the mice did not have problems with vision. There was no transgenic model Example 8 effect. All groups performed essentially the same and there 55 were no statistical differences among groups (Table 24). The Radiolabeled 'I IgG Reaches the CNS with one statistical difference came in trial 1, due to a strong Intranasal Delivery performance by WT-Sal that did not carry over into subse quent trials. There was also no evidence of a drug effect A study was conducted to determine the feasibility and to among transgenic groups (Table 24, Table 55, Table 56, 60 optimize the methods used to determine the amount of Table 57, and Table 58). However, much like with the MWM intravenously and intranasally delivered radiolabed 'I IgG hidden platform tests, there was a trend for WT-high mice to reaching the CNS in rats and mice at a two hour time point. outperform all other groups (Table 55, Table 56, Table 57, Experimental Design: There were two phases of this and Table 58). T-test comparisons between WT-Sal and experiment. In phase 1, six mice and rats were used to test WT-high for each individual trial were not significant, but a 65 a variety of different methods including anesthesia with 2 T-test for all trials between these two groups had a p-value hour Survival, drug administration methods (intravenous of 0.06. infusion through cannulations of the jugular vein in rats and US 9,556.260 B2 131 132 mice, intranasal tube method in rats), transcardial perfusion mg/kg) was used. All anesthesia was administered as Sub (with and without a non-ionic detergent), and tissue pro cutaneous injections. Boosters alternated between the Cock cessing for depletion and gamma counting. Ani tail above and 50 mg/kg Ketamine. Reflexes were tested to mals and the methods tested with each are shown in Table assess level of anesthesia every 10-15 minutes throughout 51. the study. TABLE 59 Experimental design of phase 1 of Example 8. R = rat and M = mouse. Brain Animal IV delivery IN delivery Perfusion Dissection a-R-1 Jugular Vein No infusion IN tube Saline Who e Brain Cannulation method COval a-R-2 Jugular Vein 2 mg/mL BSA IN tube 0.05% Triton X Who e Brain Cannulation until death method COval a-R-3 Jugular Vein 2 mg/mL BSA IN tube Saline Capi lary Cannulation over 1 hour method Depletion a-R-4 Jugular Vein No infusion No IN delivery 0.1% Triton X Who e Brain Cannulation COval a-R-5 Jugular Vein No infusion IN tube 0.1% Triton X Who e Brain Cannulation method COval a-R-6 Jugular Vein No infusion No IN delivery Saline Capi lary Cannulation Depletion a-M-1 Jugular Vein No infusion No IN delivery Saline Capi lary Cannulation Depletion a-M-2 Jugular Vein 2 mg/mL BSA No IN delivery 0.05% Triton X Who e Brain Cannulation over 1 hour COval a-M-3 Jugular Vein 2 mg/mL BSA No IN delivery 0.1% Triton X Who e Brain Cannulation over 1 hour COval a-M-4 Jugular Vein 2 mg/mL BSA No IN delivery Saline Who e Brain Cannulation over 1 hour COval a-M-5 Jugular Vein 8 g/kg IgG No IN delivery 0.05% Triton X Who e Brain Cannulation over 1 hour COval a-M-6 Jugular Vein 8 g/kg IgG No IN delivery 0.05% Triton X Who e Brain Cannulation over 1 hour COval

In Phase 2, three tissue processing techniques after Intranasal deliver in rats was performed using a special administration of high IVIG does in 18 rats were tested in 35 ized pipette tip. The specialized pipette tip was inserted into order to determine the optimal technique of Subsequent the rat naris. The pipette tip was created by cutting 23 mm Phase 1 experiments. The 18 rats were divided into 3 off the end of a gel loading pipette tip and attaching a 16 mm experimental groups (Table 60). length of tubing (ID=0.04 mm, OD=0.07 mm). The tubing was placed over the wide end of the pipette tip with an TABLE 60 40 overlap of 5.5 mm, and a black mark with a sharpie was made at 14.5 mm from the narrowest end of the pipette tip. Experimental groups for Phase 2. The narrow end was ultimately inserted into the rat’s nose up Group 1 Group 2 Group 3 to the black mark. For intranasal delivery, the fully anesthetized rat was 'I-IVIG dose 200 mg 200 mg 200 mg 45 Perfusion 140 mL. 140 mL saline with 90 mL saline, 25 mL. placed on its back on a heating pad in a metal Surgical tray. Saline capillary depletion 0.025% Triton X-100, The heating pad and rectal probe was used to maintain the 25 mL saline rats core temperature at 37° C. A 2"x2" gauze pad was = 6 rats 6 rats 6 rats rolled into a pillow and was securely taped. The pillow was 50 then placed under the rat’s neck to ensure that the underside Adult male Sprague Dawley rats (N=6, average weight from nostril to torso was planar and horizontal. 250 g) and adult male C57blk mice (n-6, 7-8 weeks) were A lead impregnated shield was placed between the Sur used for Phase 1. Adult male Sprague Dawley rats (N=18, gical tray and the experimenter for protection against radia average weight 264 g) were used for Phase 2. The animals tion. The dose solution, pipette, pipette tips, and waste were housed in pairs with free access to food and water and 55 receptacle were arranged behind the shield for easy access. were kept on a 12 h light cycle. The modified pipette tip was inserted into the rat naris up to Prior to commencing the Phase 1 and 2 experiments, the the black mark. The sample to be delivered (40-50 ul) was animals were allowed to normalize in the facility for a period drawn into a pipettor, the tip of the pipettor placed into the of three days before handling occurred. Animals were open tube at the end of the modified pipette tip (while slowly acclimated to human handling over a period of about 60 carefully holding the modified pipette tip in place in the rats two weeks. Enrichment food treats are given after handling nose), and then the entire dose was slowly expelled into the to encourage a human-animal bond while the acclimation rat’s nostril. process proceeds. Restraint techniques were kept brief and After the animals were euthanized, their brains were facilitated by using a towel, restraint device, or Scruffing, removed for analysis. With a large Surgical Scissors, the head when working with mice. 65 of the animal was removed by cutting dorsal to Ventral to An anesthesia cocktail containing Ketamine HCl (30 avoid contamination. Using a scalpel, the fur and skin on the mg/kg), Xylazine HCl (6 mg/kg), and Acepromazine (1 top of the skull was cut from nose to point of decapitation. US 9,556.260 B2 133 134 The skin was folded back and held with a small gauze pad enized brain sample in order to provide a final concentration to expose the top of the skull. Using a small hemostat, the of 15.5% Dextran in the homogenate. The homogenate was remainder of the spinal column was chipped away exposing then vortexted, homogenized for a second time with vertical the upper cervical spinal cord and posterior brain (cerebel , and then decanted into a small glass centrifuge tube. lum). Next, the top of the skull was removed to the olfactory The homogenate was then centrifuged in a Swinging bucket bulbs exposing the entire dorsal side of the brain. The rotor for 15 minutes at 4° C. at a speed of 5400xg. The hemostat was inserted with one blade scraping the ventral homogenate was separated into the following layers: a surface of the skull. This ensured the integrity of the dorsal bottom pellet containing the capillary segments, a clear Surface of the brain was maintained. A small spatula was liquid layer, and a top "cream layer containing the nervous used to loosen the lateral surfaces of the brain from the skull 10 tissue. Using a transfer pipette, the cream and clear liquid and dura. The brain was inverted over a clean Petri dish. The layers were transferred into new tubes. The radioactivity of optic nerve was severed, which released the brain from the the Supernatant and the pellet was determined using a skull. The brain was assessed for quality of perfusion. gamma counter. The brain was placed dorsal side up. A single edged razor Results: The data from Phase 2 shows that intravenous blade was used to sever the olfactory bulbs from the brain at 15 "I-IVIG reached the central nervous system. The animals the natural angle. Olfactory bulbs were collected. Razor with capillary depletion tissue processing had the most WIG blades were used to cut the brain into seven coronal sections in the brain tissue (49,791 ng). The animals perfused with (see FIG. 5). Each section was hemisected and placed into 0.025% Triton X as a second perfusate had the least IVIG in tubes for counting. the brain tissue (33.855 ng) (Table 61 and Table 62). The For capillary deletion, each brain section was weighed capillary depletion pellet which should hold all of the IVIG and transferred to an ice cold ground glass homogonizer. A stuck to the capillary walls only accounted for -3% of the volume of 2.857 times the tissue sample weight of buffer, pH whole brain IVIG in those animals (Table 63). The low 7.4 (10 mM HEPES, 141 mM NaCl, 4 mM KC1, 2.8 mM amount of IVIG in the capillary pellet could be a result of CaCl, 1 mM MgSO HO, 1 mM NaH2PO, and 10 mM homogenization friction during processing, releasing the D-Glucose), was added to the homogonizer. The brain 25 IVIG stuck to the capillary walls and allowing it to be mixed sample was homogenized using vertical strokes. A Small in with the Supernatant instead of staying with the volume of 26% dextran solution was added to the homog in the pellet. TABLE 61

- °I-IVIGY SPSS present IIin theSSSISYOS central nervous SYSSsystem measuredSSIS in SMCPM. Total CPM Total CPM Total CPM Total CPM Total CPM Perfusate(CPMul Rat Method Whole Brain Liquid Pellet R. Hemisphere L. Hemisphere (2nd) (3rd) b-1 Cap Dep 68,554 65,326 3,228 30,687 37,867 b-4 Cap Dep 40,791 39,372 1419 28,352 2,439 b-7 Cap Dep 29,048 28,229 819 13,374 5,674 b-10 Cap Dep 15.498 14,851 647 8,104 7,393 b-13 Cap Dep 47,908 46,533 1,376 28,757 9,151 b-16 Cap Dep 69,964 68,128 1,836 29.458 40,505 b-3 Control 98,341 52,972 45,368 278 356 b-6 Control 21,141 10,557 0,584 112 144 b-11 Control 36.457 19,077 7,380 141 121 b-15 Control 28,303 14,228 4,075 126 66 b-17 Control 20,524 9,508 1,016 231 127 b-18 Control 38,683 19,350 9,333 125 73 b-2 Triton X 36,984 16,622 20,362 S4O 216 b-5 Triton X 49,882 25,617 24,264 98 219 b-8 Triton X 19,194 11,031 8,163 243 no sample b-9 Triton X 33,716 15,026 8,690 422 82 b-12 Triton X 21,255 7,639 3,616 527 151 b-14 Triton X 14,013 6,712 7,301 441 117 Average Cap Dep 45,294 43,740 1,554 23,122 22,172 Average Control 40,575 20,949 9,626 169 148 Average Triton X 29,174 13,775 5,399 379 157

TABLE 62 ng by Group Total ng Total ng Total ng Total ng Total ng Perfusate(ngul Rat Method Whole Brain Liquid Pellet R. Hemisphere L. Hemisphere (2nd) (3rd) 1b-1 Cap Dep 68,537 65,310 3,227 30,679 37,858 1b-4 Cap Dep 45,383 43,804. 1579 31,544 13,840 1b-7 Cap Dep 32,060 31,156 904 14,761 17,300 1b-10 Cap Dep 18,231 17470 761 9,534 8,697 1b-13 Cap Dep 57,258 55,614 1,644 34,369 22,889 1b-16 Cap Dep 77.276 75,248 2,028 32,537 44,739 1b-3 Control 108.404 58,393 50,011 306 392 US 9,556.260 B2 135 136 TABLE 62-continued ng by Group Total ng Total ng Total ng Total ng Total ng Perfusate(ngful Rat Method Whole Brain Liquid Pellet R. Hemisphere L. Hemisphere (2nd) (3rd) 1b-6 Control 24,824 12,397 12,428 132 169 1b-11 Control 35,411 18,530 16,881 137 118 1b-15 Control 36,686 18,442 18,244 163 86 1b-17 Control 25,940 12,017 13,923 292 160 1b-18 Control 50,757 25,390 25,367 16S 95 1b-2 Triton X 46,547 20,921 25,626 68O 272 1b-5 Triton X 56,294 28,910 27,383 111 247 1b-8 Triton X 22,577 12,975 9,601 285 no sample 1b-9 Triton X 39,032 17,396 21,637 488 95 1b-12 Triton X 22,099 7,943 14,157 S48 157 1b-14 Triton X 16,581 7.942 8,639 522 138 Average Cap Dep 49,791 48,101 1,690 25,571 24.220 Average Control 47,004 24,195 22,809 199 170 Average Triton X 33,855 16,014 17,841 439 182

TABLE 63 ng by Group

Percent Percent of Percent of est. of ng Percent Percent of ng ng est. ng in est. ng in Percent delivered of whole whole delivered delivered est. ng in 2nd 3rd ng in (Whole brain brain (2nd (3rd blood perfusate perfusate blood Brain) (Liquid) (Pellet) perfusate) perfusate) Average 124,564,379 62% O.02% 97% 3% Cap Dep Average 151,853,766 4,978,470 4.249,634 76% O.O.2% 2.5% 2.1% Control Average 134,662,521 10,980,039 4,543,372 67% O.02% 5.5% 2.3% Triton X *The total estimated blood volume was determined as the body weight times 0.06 plus 0.77 (Lee and Blaufox, 1985).

The Triton X perfusion methods resulted in a 28% reduc group. An increase in the total volume of perfusate in the tion of IVIG whole brain concentration versus the saline next Phase should solve this issue. perfusion control. The perfusate should show the amount of 40 IVIG cleared from the blood vessels over the course of the Example 9 25 ml (perfused at a rate of 15 ml/min). Three 250 LA samples of each perfusate were counted in the gamma counter. Averages of the three were than calculated. To Biodistribution of IgG administered Intranasally determine the total amount of IVIG in each perfusate, the 45 and Intravenously in Mice ng/ul IVIG concentration was determined and multiplied by 25000 (the 25 ml of perfusate used). The first perfusates A study was conducted to compare the biodistribution of (-90 ml at 15 ml/min) were not collected since this step was pooled human immunoglobulin G (IgG) administered to the same in all of the animals in the study. In the group 50 mice intranasally and intravenously. Delivery of IgG to the perfused with 0.025% Triton X, more ''I-IVIG was brain and residual IgG in the bloodstream were determined. removed (439 ng/ul) than the groups perfused with saline (199ng/ul). This difference was not seen in the 3" perfusate, Experimental Design: IgG radiolabeled with iodine-125 meant to clear any remaining Triton X from the blood (*I-IgG) was either infused into the left femoral vein vessels, (170 ng/ul and 182 ng/ul, respectively) (Tables 1 55 (intravenous administration; IV) or intranasally adminis and 2) Suggesting that the maximum clearance of IVIG from tered (IN) as drops to anesthetized rats over 14 minutes. the vessels at this concentration of Triton X was achieved. Animals were sacrificed and concentrations of I-IgG were A higher Triton X concentration in the perfusate may yield determined in the brain, blood, and body of the mice at nine a further reduction. different time points (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 In these results, approximately 0.02% of the total deliv 60 h, 24 h, and 72 h post-IgG administration). Blood samples were taken from the heart, animals were perfused, and brains ered IVIG that was infused reached the brain (Table 55) in removed. Radiolabeled IgG was detected with a gamma all methods. During the Phase 2 experiments it was noted counter for quantitative analysis. Half of each brain was that the brain tissues were slightly pinkish, Suggesting the processed into Supernatant and run through a size exclusion total Volume perfused was not adequate to completely 65 column to explore intactness of the I-label. The three remove blood from the brain. This slight coloration appeared experimental cohorts were administered IgG as described in consistent throughout all animals in each experimental Table 64. US 9,556.260 B2 137 138 TABLE 64 Animals were then monitored for adverse effects and anes thesia levels until the euthanasia time point was reached. Treatment groups assigned for intranasal administration of IgG. During intranasal delivery, a 500 ul sample of saline was Cohort IgG Dosage Administration infused over 14 minutes through the left femoral vein. All animals were rolled off of their backs at 15 min after the Intranasal Drops - High 0.02 g/kg one drop every 2 minutes to Dose (IN Drop - high) alternating naris; infusion of completion of delivery. Saline For IV delivery, anesthetized animals were placed on their Intranasal Drops - Low 0.002 g/kg one drop every 2 minutes to Dose (IN Drop - low) alternating naris; infusion of backs. A blunt 22 gauge needle attached to a 1 cc syringe Saline 10 was inserted into the femoral vein canula. "I-IgG was Intranasal Device - 0.02 g/kg two puffs to alternating naris at prepared in 500 ul aliquots and infused over 14 minutes. (IN Device) O and 10 minutes, with Animals were then monitored for adverse effects and anes accompanying control intravenous infusion of Saline thesia levels until the euthanasia time point was reached. Intravenous (IV) 0.02 g/kg infusion to left femoral vein over At the experimental end time, blood was drawn directly fourteen minutes 15 from the heart and animals were perfused with 120 ml ice *3 ratsitime point for a total of 27 rats per experimental group cold saline directly through the heart. One small drop of blood was placed into a pre-weighed, labeled gamma tube On the day of delivery, each 'I-IgG aliquot was and approximately 0.6 mL was placed into a labeled serum removed from the freezer and allowed to come to room separator tube. The serum separator tube was spun and temperature (about 20 minutes). The aliquots were then serum was collected. The serum was diluted in homogeni gently vortexed. A sample of 1 Jul was placed into 999 ul of zation buffer. The diluted serum was further spun down in a water and vortexed (1:1,000 dilution). Three 20 Jul samples 100 kDa size exclusion filtration device. Samples were were removed from the dilution and placed into labeled collected from both the top of the filter and the bottom and gamma tubes. An additional 10 ul was placed into 90 ul of 25 placed into labeled gamma tubes. The filter was also col water and vortexed (1:10,000 dilution). Three 20 ul samples lected and placed into a labeled gamma tube. were removed from the 1:10,000 dilution and placed into The brain was extracted from the skull and hemisected. labeled gamma tubes. Standards were later quantified The left hemisphere was further processed as described through gamma counting. All doses within groups were below. The right hemisphere was weighed, cut into 7 pieces equalized for Volume, weight (mg), and radioactivity (LLCi) 30 and placed into labeled gamma tubes. by varying the dilution with saline to account for the decay Additionally, the olfactory and respiratory epithelia were of 125I. collected separately. The epithelia were expected to contain Adult male Sprague-Dawley rats (Animal Care Facility at higher amounts of 'I than the quantitation limit of the Regions Hospital from Harlan) with the left femoral vein 35 gamma counter, so both were split into multiple pieces. Each canulated were used in this experiment. All rats weighed piece of epithelia was placed into a pre-weighed, labeled approximately 250 g to ensure accurate dosing. The animals gamma tube. were housed individually with free access to food and water. The left hemisphere was weighed after removal from the Animals were kept on a 12-hour light cycle. skull. It was homogenized and spun down to retrieve Super For the IN Drop, IN Device, and IV administrations, an 40 natant. The supernatant was further spun down in a 100 kDa anesthesia cocktail containing ketamine HCl (30 mg/kg), size exclusion filtration device. Samples were collected from Xylazine HCl (6 mg/kg), and acepromazine (1 mg/kg) was both the top of the filter and the bottom and placed into used. All anesthesia was administered as Subcutaneous labeled gamma tubes. The pellet was collected and placed injections. Boosters alternated between the cocktail into a pre-weighed labeled gamma tube. The filter was also described above and 50 mg/kg ketamine. Reflexes were 45 collected and placed into a labeled gamma tube. tested to assess level of anesthesia every 10-15 minutes 3-5 mm samples of body tissues were collected and throughout the study. Animals in groups sacrificed at 4 hr placed into pre-weighed, labeled gamma tubes. Body tissues and beyond were allowed to recover from anesthesia and include: liver, spleen, kidney, Small intestine, lung, esopha were re-anesthetized prior to euthanasia. 50 gus, trachea, and blood (drawn directly from the heart as For IN Drop delivery, anesthetized rats were placed on described above). The gamma tubes containing samples their backs on a heating pad. 'I-IgG was administered were counted using a COBRA II Auto-Gamma Counter. intranasally as 8x6 uL nose drops with an Eppendorf Results: Intactness of IgG in the brain was slightly less pipettor to alternating nares every 2 minutes for a total with intranasal administrations (example: IN high 49%, IN volume of 48 uL. Animals were then monitored for adverse 55 low 49%, IN device 40% at 15 minutes) as compared to effects and anesthesia levels until the euthanasia time point was reached. During intranasal delivery, a 500 uL sample of intravenous administration (69% at 15 minutes) in the earlier saline was infused over 14 minutes through the left femoral time points (Table 65, Table 66, Table 67, and Table 68). vein. All animals were rolled off of their backs at 15 minutes However, because of the non-validated method of calculat 60 ing the intactness and the limitations of the gamma counting after the completion of delivery. machine, non-intact or “free” 'I may be magnified. The For IN Device delivery, anesthetized animals were placed CPM counts from gamma tubes for aliquots representing the on their backs and a tube was inserted about 14 mm deep “bottom’ of the filtration device tubes (where the non-intact into the nostril. The tube was connected to an actuator that IgG would be expected) were rather low in many of IN delivered 15 uL of dosing solution toward the olfactory 65 treated animals. It is usually desired that the counts reach at epithelium. One bolus was sprayed at the start of delivery, least two times background (in this study would be ~50 one was sprayed at 10 minutes after the onset of delivery. CPM). US 9,556.260 B2 139 140 TABLE 65 Biodistribution and intactness of IgG administered to rats via high dose nasal drops 0.02 g IgG/kg). ugg IN-Drops High

IN IN IN IN IN IN IN IN IN High High High High High High High High High Time

15 min 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 72 hr Raw ugg Dosed ugg 60 uCi 92,625,403 99,889,203 97,886,218 111,043,619 101,049,672 99,398,932 78,063,258 108,114,877 76,689,700 Total ugg

Olfactory 585 127 120 938 167 118 2O 10 O.S6 Epithelium Respiratory 8,614 11,790 13,222 16,686 5,189 1,312 41 10 2.4 Epithelium R. Hemisphere O.24 O.22 O.18 O.10 O.11 O.15 O.22 O16 O.O39 L. Hemisphere O.11 O.272 O.200 O.126 O.095 O.128 O.231 O.145 O.O29 (total recovered) Dosing Solution 38,594 41,621 40,786 46,268 42,104 41-416 32,526 45,048 31,954 (1:1,000) ugg

Blood 3.1 3.3 4.4 4.0 3.7 5.3 7.3 5.4 O.8 Liver O.23 O46 O.S1 O43 0.44 1.O O.9 1.1 O.24 Spleen 0.55 1.1 1.4 1.2 1.4 1.2 2.4 1.5 O16 Kidney O.9 1.9 2.7 1.5 1.6 2.8 3.8 2.2 O.39 Small Intestines O.32 0.4 O.91 0.75 2.2 2.5 6.3 1.3 O.09 Lung O.9 1.8 1.5 1.O 1.3 2.1 2.4 1.7 O.26 Esophagus O.S1 O.61 1.1 O.9 33 126 4.9 6 O.22 Trachea 0.75 0.77 4.0 1.7 3.0 2.4 17 5 O.25 Intactness

N1 Brain 49% 46% 40% 48% 51% 53% 49% 49% 66% N1 Blood 39% 32% 35% 33% 16% 27% 30% 27% 54%

TABLE 66 Biodistribution and intactness of IgG administered to rats via low dose nasal drops 0.002 g IgG/kg). lugg. IN Drops-LOW

IN Low IN Low IN Low IN Low IN Low IN Low IN Low IN Low IN Low Time

15 min 30 min 1 hr 2 hr 4 hr 8 hr 12 hr 24 hr 72 hr Dosed ugg (60 uCi 91,152,030 71,046,179 83,024,122 109,042,942 102.934,060 64,471,560 78,549,717 72,139,899 64,456,268 Total ugg

Olfactory 118 57.6 58.7 58.0 56 1.9 25.9 6.69 0.571 Epithelium Respiratory 9,930 12,284 10.402 6,716 3,055 101 111 7.8 1.2O Epithelium R. Hemisphere O.O60 O.048 O.O31 O.O2O O.O15 O.O26 O.O32 O.O15 O.OO44 L. Hemisphere 0.057 O.042 O.O23 O.018 O.O16 O.O27 O.O3O O.O14 O.OO40 (total recovered) Dosing Solution 37,980 29,602 34,593 45,435 42,889 26,863 32,729 30,058 26,856 (1:1,000) ugg

Blood O41 O.S6 O.S1 0.44 0.37 O.78 O.99 0.57 OO67 Liver O.091 O.09 O.O6 O.O86 O.061 O.15 O.19 O.12 O.036 Spleen O.15 O.21 O.31 O.19 O.12 O.38 O.30 O.20 O.O23 Kidney O.22 O.26 0.27 O.1 O.20 O.S3 O.63 O.28 O.042 Small Intestines 0.075 O16 O.10 O.13 O.18 O.33 O.29 O.OS8 O.O12 Lung O.14 O.25 O.09 O.15 O.17 O.26 O.43 O.29 O.O32