Ex Parte Mendoza et alDownload PDFPatent Trial and Appeal BoardJan 12, 201512647971 (P.T.A.B. Jan. 12, 2015) Copy Citation UNITED STATES PATENT AND TRADEMARK OFFICE __________ BEFORE THE PATENT TRIAL AND APPEAL BOARD __________ Ex parte ALBERTO L. MENDOZA, ROBERT L. GLASS, and RICHARD D. HANSEN __________ Appeal 2012-007323 Application 12/647,971 Technology Center 1600 __________ Before ERIC B. GRIMES, JEFFREY N. FREDMAN, and ULRIKE W. JENKS, Administrative Patent Judges. FREDMAN, Administrative Patent Judge. DECISION ON APPEAL This is an appeal1 under 35 U.S.C. § 134 involving claims to a method of treating diseases not caused by Pythium insidiosum. The Examiner rejected the claims as failing to comply with the enablement requirement. We have jurisdiction under 35 U.S.C. § 6(b). We affirm and designate our affirmance as a New Ground of Rejection pursuant to our authority under 37 C.F.R. § 41.50(b). 1 Appellants identify the Real Party in Interest as Michigan State University Board of Trustees (see App. Br. 1). Appeal 2012-007323 Application 12/647,971 2 Statement of the Case Background “The present invention has as its primary objective both widening the number of species that can be treated with P. insidiosum protein and widening the scope of diseases that can be effectively treated by modulating the immune response in an animal” (Spec. 1, ll. 24–26). The Claims Claims 1–6 are on appeal.2 Independent claim 1 is representative and reads as follows: 1. A method of treating diseases not caused by Pythium insidiosum (P. insidiosum), comprising: injecting into an animal not suffering from P. insidiosum infection or allergy, to treat diseases not caused by P. insidiosum, proteins expressed from P. insidiosum MTPI-04, ATCC-PTA-12166 to generate a therapeutic response within the animal. The issue3 The Examiner rejected claims 1–6 under 35 U.S.C. § 112, first paragraph as failing to comply with the enablement requirement (Ans. 5–6; Final Rej. 6–23). The Examiner finds that [T]he specification, while being enabling for a method of reducing the size of lesions in a patient with chromoblastomycosis fungal infection and a method for the 2 Claims 7–12 were withdrawn from consideration and subsequently canceled (see App. Br. 3). 3 The Examiner has withdrawn the written description, deposit, and indefiniteness rejections (see Ans. 4). Appeal 2012-007323 Application 12/647,971 3 reduction of lesions associated with equine sarcoids comprising injecting into said animal which does not appear to be suffering from Pythium insidiosum infection or allergy proteins expressed from Pythium insidiosum, does not reasonably provide enablement for a method of treating diseases not caused by P. insidiosum, comprising: injecting into an animal not suffering from P. insidiosum infection or allergy, to treat diseases not caused by P. insidiosum, proteins expressed from P. insidiosum MTPI-04, ATCC- PTA-12166 to generate a therapeutic response within the animal. (Ans. 5–6.) The Examiner finds the Specification is not “enabling for one skilled in the art to make and use the claimed invention as the amount of experimentation required is undue, due to the broad scope of the claims, the lack of guidance and working examples provided in the specification and the high degree of unpredictability” (Final Rej. 23). The issue with respect to this rejection is: Does the evidence of record support the Examiner’s conclusion that the claims fail to comply with the scope of enablement requirement? Findings of Fact The following findings of fact (“FF”) are supported by a preponderance of the evidence of record. Breadth of Claims 1. The Examiner finds that the “claims are broad and encompass treating any disease that is not caused by P. insidiosum” (Final Rej. 19). Appeal 2012-007323 Application 12/647,971 4 2. Olsen4 teaches examples of immunologic deficiency syndromes not caused by P. insidiosum which: [I]nclude, but are not limited to: blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria. (Olsen, col. 96, ll. 36–45.) 3. Olsen teaches examples of autoimmune disorders which: [I]nclude, but are not limited to: Addison’s Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture’s Syndrome, Graves’ Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter’s Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease. (Olsen, col. 97, ll. 8–18.) 4. Olsen teaches examples of cardiovascular disorders which: [I]nclude cardiovascular abnormalities, such as arterio- arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, 4 Olsen et al., US 6,632,920 B1, issued Oct. 14, 2003. Appeal 2012-007323 Application 12/647,971 5 crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein’s anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher’s Syndrome, trilogy of Fallot, ventricular heart septal defects. (Olsen, col. 98, ll. 29–42.) 5. Olsen teaches examples of cancers including [F]ollicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi’s sarcoma and ovarian cancer. (Olsen, col. 101, ll. 46–55.) 6. Olsen teaches examples of neurodegenerative disorders including “Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease” (Olsen, col. 102, ll. 36–39). 7. Olsen teaches examples of infectious diseases including [H]epatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt’s Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi’s, warts), and viremia . . . bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, Appeal 2012-007323 Application 12/647,971 6 uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter’s Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat- Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections . . . Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis. (Olsen, col. 105, l. 16 to col. 106, l. 5.) Presence of Working Examples 8. The Specification provides twelve examples, in which individual patients or animals were treated with the Pythium Immunotherapeutic product (see Spec. 6–10). 9. None of the examples in the Specification incorporate negative controls to demonstrate comparative efficacy of the Pythium Immunotherapeutic product (see Spec. 6–10). 10. The examples do not provide the natural history of the diseases, or indicate the frequency of spontaneous resolution. In Example 3, the lesion of a single treated wounded horse healed and the horse grew a thick winter coat (see Spec. 7, ll. 20–25). Example 3 does not indicate how long such a lesion would normally require to heal on its own, nor does the example provide any evidence regarding the likelihood of thick winter coats. None of the examples, including Example 3, provide any controlled Appeal 2012-007323 Application 12/647,971 7 experimental evidence showing that the Pythium Immunotherapeutic product was responsible for the treatment result in any way. Amount of Direction or Guidance Presented 11. The Specification teaches: [P]ythium immunotherapy demonstrated benefit for treatment of, or managing diseases other than caused by Pythium insidiosum, such as for example: Sarcoid (Equine); Mast cell tumor (Canine); Allergic Disease (Canine, Feline, Equine, Human); Inflammatory Bowel Disease (Canine); Miliary dermatitis (Feline); Exuberant Granulation (Equine); Chromoblastomycosis (Human); Asthma (Feline); Otitis Externa (Canine, Feline); Arthritis (Canine); Anhidrosis (Equine); and Navicular Disease and Laminitis (Equine). (Spec. 2, ll. 17–22.) 12. The Specification teaches a process for preparing a Pythium culture extract and teaches that the “immunotherapeutic concentrate contains between about 20 mcg to 5.0 mg of protein per dose. The immunotherapeutic dosage preferred for some animals is between about 20 mcg/mL and 40 mcg/mL” (Spec. 3, l. 28 to Spec. 6, l. 7). State of the Prior Art and Unpredictability of the Art 13. Ernst5 teaches that: It is, of course, tempting to assume a causal relationship where only a temporal link exists between an intervention and a clinical outcome. Throughout history, medical progress has been hindered by this misapprehension. But medicine has moved on, and we now 5 Ernst, E., How the public is being misled about complementary/ alternative medicine, 101 J. ROYAL SOCIETY MEDICINE 528–530 (2008). Appeal 2012-007323 Application 12/647,971 8 know (or should know) that millions can be entirely wrong when assuming that this or that treatment ‘has worked for them’. Clinical improvement can be due to a host of factors other than the specific effect of a treatment, yet the anecdote remains perhaps the most effective method by which the press and others mislead the public. (Ernst 529, col. 1 to 2.) 14. Chambers6 teaches that “[e]fficacy of different treatments is difficult to assess because most studies have not been controlled” (Chambers 1059, col. 2). 15. Chambers teaches that “many private practitioners treat sarcoids successfully by a policy of non-intervention, which again may represent a specific population of sarcoids that remain quiescent or the rare spontaneous regressors and there is some anecdotal evidence for this” (Chambers 1059, col. 2). 16. Bertone7 teaches that “[a]ttempts to treat or prevent exuberant granulation tissue have probably been made with every possible chemical substance, physical contraption, and surgical procedure known or imagined. Controlled research evaluating most of these treatment regimens is lacking or contradictory” (Bertone 554). 6 Chambers et al., Association of bovine papillomavirus with the equine sarcoid, 84 J. GENERAL VIROLOGY 1055–1062 (2003). 7 Bertone, A., Management of Exuberant Granulation Tissue, 5 THE VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE 551–562 (1989). Appeal 2012-007323 Application 12/647,971 9 17. Gershell8 teaches that “[a]ll too frequently, a compound that works well in the laboratory and in preclinical trials, and even in small patient samples, meets with disaster when applied to larger study groups” (Gershell 326, col. 2). 18. Hann9 teaches that “[m]edicinal Chemistry is at the heart of the drug discovery process. . . . This process more often resembles an unpredictable journey on a chaotic surface rather than a quantitative and predictive science” (Hann 349, col. 2). 19. Laohapensang10 teaches that “[i]mmunotherapy using P. insidiosum antigen (PIA) may be effective in treating human pythiosis” (Laohapensang 123, col. 1). Quantity of Experimentation 20. The Examiner finds that the “quantity of experimentation necessary would be undue to arrive at the method as claimed. Reasonable correlation must exist between the scope of the claims and scope of enablement set forth, and it cannot be predicted from the disclosure how to make/use the claimed genus” (Ans. 22–23). 21. Kaitin11 teaches that “the average time to bring a new pharmaceutical product to market, from the start of clinical testing to FDA 8 Leland J. Gershell & Joshua H. Atkins, A brief history of novel drug discovery technologies, 2 NATURE REVIEWS 321–327 (2003). 9 Hann, M., Molecular obesity, potency and other addictions in drug discovery, 2 MED. CHEM. COMMUN. 349–355 (2011). 10 Laohapensang et al., Necrotizing Arteritis Due to Emerging Pythium insidiosum Infection in Patients With Thalassemia: Rapid Diagnosis with PCR and Serological Tests — Case Reports, 14 INTERNATIONAL J. ANGIOLOGY 123–128 (2005). Appeal 2012-007323 Application 12/647,971 10 approval, is 8.5 years, and the clinical success rate is 21.5%” (Kaitin 210, col. 2). 22. Kaitin teaches that the “average capitalized cost to bring one new biopharmaceutical product to market, including the cost of failures, is $1.24 billion in 2005 dollars” (Kaitin 210, col. 1). 23. Hann teaches that the “road to the successful discovery of new medicines continues to frustrate all those involved. . . . Researchers are equally frustrated in that, despite all the accumulated knowledge from the new technologies, the challenge of successfully navigating through it to find novel drugs seems to get harder rather than easier” (Hann 349, col. 1). Skill in the Art 24. The Examiner makes no finding regarding the skill level in the art. Principles of Law Factors to be considered in determining whether a disclosure would require undue experimentation . . . include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. In re Wands, 858 F.2d 731, 737 (Fed. Cir. 1988). 11 Kaitin, K., Obstacles and Opportunities in New Drug Development, 83 NATURE 210–212 (2008). Appeal 2012-007323 Application 12/647,971 11 Analysis The analytical framework for determining whether claims fail to satisfy the enablement requirement balances the Wands factors to determine if undue experimentation would have been required to perform the reasonable scope of the claimed method at the time of filing of the Specification. We begin with claim breadth, where claim 1 encompasses any disease not caused by Pythium insidiosum (FF 1). Applying the broadest reasonable interpretation, we interpret claim 1 to encompass the full range of human diseases, except those caused by Pythium insidiosum, including immunological conditions from ataxia telangiectasia to Wiskott-Aldrich Disorder (FF2), autoimmune diseases from Addison’s Disease to Reiter’s Disease (FF 3), cardiovascular disorders from arteriovenous fistula to ventricular heart septal defects (FF 4), cancers from adenoma to prostate cancer (FF 5), neurodegenerative disorders from Alzheimer’s disease to retinitis pigmentosa (FF 6), infectious diseases from AIDS to warts (FF 7), and thousands of more disease conditions. The factor of claim breadth weighs heavily against the full scope of enablement of claim 1. While the Specification includes twelve Examples, each of these examples represents anecdotal results with no controlled evidence showing that the Pythium insidiosum product was responsible in any way for amelioration of the disease, rather than some other, unrelated reason (FF 8– Appeal 2012-007323 Application 12/647,971 12 10). We have considered the Mendoza Declaration,12 but Dr. Mendoza does not provide any controlled research studies or evidence demonstrating effectiveness of the Pythium insidiosum product. Instead, Dr. Mendoza explains that the treated animals were not infected with Pythium insidiosum (see, e.g., Mendoza Decl. 3–4). Importantly, the asserted working examples represent a very small, and non-representative fraction of the total number of diseases which fall within the scope of claim 1. See Pharm. Res., Inc. v. Roxane Labs., Inc., 253 Fed. Appx. 26, 30 (Fed. Cir. 2007) (“Given the highly unpredictable nature of the invention and the extremely broad scope of the claims, these three working examples do not provide an enabling disclosure commensurate with the entire scope of the claims.”) The factor of working examples is, at best, neutral relative to the scope of enablement of claim 1. The Specification provides guidance on making the Pythium insidiosum product (FF 12) and provides a relatively diverse list of disease targets (FF 11), but provides no guidance explaining why the Pythium insidiosum product would be expected to treat the full range of diseases within the scope of claim 1, including conditions such a congenital heart defects (FF 4), neurodegenerative disorders such as Alzheimer’s disease (FF 6) and thousands of other conditions. The factor of guidance presented weighs heavily against the full scope of enablement of claim 1. 12 Declaration of Dr. Leonel Mendoza, filed July 19, 2011. Appeal 2012-007323 Application 12/647,971 13 The prior art recognizes that anecdotal evidence is of little value and may be entirely misleading (FF 13–14). Even when therapeutic effect is seen, the prior art recognizes that this effect may be due to spontaneous regression or represent the natural course of the disease (FF 15). The prior art recognizes that the efficacy of a drug compound on a single disease, much less drug compound efficacy for the full scope of every disease which afflicts humans or other mammals, is entirely unpredictable without controlled studies demonstrating success (FF 16–17). Hann explains this unpredictability, teaching that “[m]edicinal Chemistry is at the heart of the drug discovery process. . . . This process more often resembles an unpredictable journey on a chaotic surface rather than a quantitative and predictive science” (Hann 349, col. 2; FF 18). The only disease for which the Pythium insidiosum product has shown repeated efficacy is in treatment Pythium insidiosum infection itself (see Mendoza Decl. 2 ¶ 5; FF 19). The factor of unpredictability of the art weighs heavily against the full scope of enablement of claim 1. The quantity of experimentation necessary to demonstrate the efficacy of the Pythium insidiosum product for treatment of a single disease would be substantial, with the average pharmaceutical requiring eight years of testing (FF 21) and more than one billion dollars (FF 22) with a clinical success rate of 21% for the average pharmaceutical (FF 21). Hann teaches that “the challenge of successfully navigating through it to find novel drugs seems to get harder rather than easier” (Hann 349, col. 1; FF 23). The factor of quantity of experimentation weighs heavily against the full scope of enablement of claim 1. Appeal 2012-007323 Application 12/647,971 14 There is no finding regarding the skill level in the art, so this factor remains neutral or even positive towards the full scope of enablement of claim 1. Therefore, as we balance the factors relating to enablement, including the extremely broad claims, the anecdotal nature of the twelve working examples, the absence of guidance regarding the full scope of the claim in the Specification, the extremely high level of unpredictability in the art, the immensely large quantity of experimentation required to practice the full scope of the claim, with the high skill level in the art, we conclude that the balance of the Wands factors supports the Examiner’s finding that the claimed invention fails to comply with the scope of enablement requirement. Appellants contend that the “Examiner basically refuses to accept the Declaration of Dr. Mendoza and second guesses the science of examples 3- 12” (App. Br. 7–8). We are not persuaded. The Examiner properly assessed the Wands factors, recognizing the breadth of claim 1 is not limited to examples 1–12, but rather encompasses a method of treating any diseases not caused by Pythium insidiosum (Ans. 8). Appellants, the Specification, and the Mendoza Declaration do not provide enabling support for the full breadth of claim 1 as discussed above. The Examiner has acknowledged that undue experimentation would not have been required to perform the treatments of the two specific diseases of examples 1 and 2. However, with regard to the remaining examples 3–12, these anecdotal treatments fail the basic requirement of scientific reproducibility, lacking any showing that even a second animal was successfully treated for the same condition, much less Appeal 2012-007323 Application 12/647,971 15 that the improvement was due to the Pythium insidiosum product and does not represent the natural history and resolution of the particular diseases. Appellants contend that “Dr. Mendoza points out the supporting evidence and why the Examiner’s conclusions as expressed in the first Office Action are either scientifically incorrect or are based on assumptions of fact that are wrong” (App. Br. 8). We are not persuaded. We accepted Dr. Mendoza’s finding that the diseases listed in examples 3–12 are not caused by Pythium insidiosum. However, Appellants’ argument over whether the diseases listed in examples 3–12 are allergic in nature fails to address the central enablement concerns in this case regarding the very broad scope of the claim, the absence of a commensurate number of working examples, the anecdotal nature of those working examples, the absence of guidance regarding the full claim scope in the Specification, the unpredictability of disease treatment, and the very large quantity of experimentation (FF 1–23). Appellants make no effort to demonstrate enablement regarding these Wands factors. Conclusion of Law The evidence of record supports the Examiner’s conclusion that the claims fail to comply with the scope of enablement requirement. SUMMARY In summary, we affirm the rejection of claim 1 under 35 U.S.C. § 112, first paragraph as failing to comply with the enablement requirement. Pursuant to 37 C.F.R. § 41.37(c), claims 2–6 fall with claim 1, as these claims were not argued separately. Appeal 2012-007323 Application 12/647,971 16 Because our reasoning and evidence differs from that of the Examiner, we designate the affirmance as a new ground of rejection pursuant to 37 C.F.R. § 41.50(b) (effective September 13, 2004, 69 Fed. Reg. 49960 (August 12, 2004), 1286 Off. Gaz. Pat. Office 21 (September 7, 2004)). 37 C.F.R. § 41.50(b) provides “[a] new ground of rejection pursuant to this paragraph shall not be considered final for judicial review.” 37 C.F.R. § 41.50(b) also provides that the Appellants, WITHIN TWO MONTHS FROM THE DATE OF THE DECISION, must exercise one of the following two options with respect to the new ground of rejection to avoid termination of the appeal as to the rejected claims: (1) Reopen prosecution. Submit an appropriate amendment of the claims so rejected or new evidence relating to the claims so rejected, or both, and have the matter reconsidered by the Examiner, in which event the proceeding will be remanded to the Examiner. . . . (2) Request rehearing. Request that the proceeding be reheard under § 41.52 by the Board upon the same record. . . No time period for taking any subsequent action in connection with this appeal may be extended under 37 C.F.R. § 1.136(a). AFFIRMED; 37 C.F.R. § 41.50(b) cdc Notice of References Cited Application/Control No. 12/647,971 Applicant(s)/Patent Under Reexamination Alberto L. Mendoza et al. Examiner Lakia Tongue Art Unit 1600 Page 1 of 1 U.S. PATENT DOCUMENTS * DOCUMENT NO. DATE NAME CLASS SUBCLASS DOCUMENT SOURCE ** APS OTHER A 6,632,920 10/14/03 Olsen et al. B C D E F G H I J K L M FOREIGN PATENT DOCUMENTS * DOCUMENT NO. DATE COUNTRY NAME CLASS SUBCLASS DOCUMENT SOURCE ** APS OTHER N O P Q R S T NON-PATENT DOCUMENTS * DOCUMENT (Including Author, Title Date, Source, and Pertinent Pages) DOCUMENT SOURCE ** APS OTHER U Ernst, E., How the public is being misled about complementary/ alternative medicine, 101 J. ROYAL SOCIETY MEDICINE 528–530 (2008). V Leland J. Gershell & Joshua H. Atkins, A brief history of novel drug discovery technologies, 2 NATURE REVIEWS 321–327 (2003). W Hann, M., Molecular obesity, potency and other addictions in drug discovery, 2 MED. CHEM. COMMUN. 349–355 (2011). X Kaitin, K., Obstacles and Opportunities in New Drug Development, 83 NATURE 210–212 (2008). *A copy of this reference is not being furnished with this Office action. (See Manual of Patent Examining Procedure, Section 707.05(a).) **APS encompasses any electronic search i.e. text, image, and Commercial Databases. U.S. Patent and Trademark Office PTO-892 (Rev. 03-98Notice of References Cited Part of Paper No. 16 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 93 ing concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et aI., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, aden- ovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1- antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld 94 contain the polynucleotide construct integrated into its genome, and will express the desired gene product. Another method of gene therapy involves operably asso- ciating heterologous control regions and endogenous poly- nucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; Inter- national Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, pub- et aI., Science, 252:431-434 (1991); Rosenfeld et aI., Cell, 68:143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)). 10 lished Aug. 4, 1994; Koller et aI., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijistra et aI., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower 15 level than desired. Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et aI., Cell, 68:143-155 (1992); Engelhardt et aI., Human Genet. Ther., 4:759-769 (1993); Yang et aI., Nature Genet., 7:362-369 (1994); Wilson et aI., Nature, 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus 20 vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and consti- tutively express Ela and Elb, which cOll1plell1ent the defec- tive adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of 25 adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention. Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to 30 form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, for example, the HARP promoter of the present invention, but cannot repli- cate in most cells. Replication deficient adenoviruses may be 35 deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5. In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require 40 helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space 45 for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354, 678,5,436,146,5,474,935,5,478,745, and 5,589,377. Polynucleotide constructs are made, using standard tech- niques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promot- ers are described herein. The targeting sequence is suffi- ciently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5' end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be ampli- fied using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5'and 3' ends. Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter. The amplified promoter and target- ing sequences are digested and ligated together. The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, pre- cipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below. The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endog- enous sequence is placed under the control of the promoter. 50 The promoter then drives the expression of the endogenous For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integra- tion. The polynucleotide construct containing polynucle- otides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook 55 et aI., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinantAAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate 60 precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or her- pes viruses. Once the packaging cells are transfected and infected, they will produce infectious AA V viral particles which contain the polynucleotide construct of the invention. 65 These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will sequence. The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucle- otides encoding other angiongenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase. Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the poly- nucleotide to be expressed towards or at the 5' end of the 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 95 coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically syn- thesized using methods known in the art. Any mode of administration of any of the above- described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules 96 mals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly Biological Activities The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucle- otides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter 10 infusion, biolistic injectors, particle accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially avail- able depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during 15 surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease. Immune Activity The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating deficiencies or disorders of the immune system, by activat- ing or inhibiting the proliferation, differentiation, or mobi- 1ization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers. (Kaneda et aI., Science, 243:375 (1989». A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention cOll1plexed with a delivery vehicle is adnlinistered 20 macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or son1e autoinl111ulle disorders, acquired (e.g., by by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of 25 arteries refers to injecting the composition centimeters and preferably, millimeters within arteries. chemotherapy or toxins), or infectious. Moreover, a poly- nucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder. Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be inj ected into areas of tissue inside the wound. Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes compris- ing ligands for targeting the vehicle to a particular site. Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et aI., Proc. Natl. Acad. Sci. USA, 189:11277-11281(1992), which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucle- otide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin. Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucle- otide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian. Therapeutic composi- tions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mam- A polynucleotides or polypeptides, or agonists or antago- nists of the present invention may be useful in treating or 30 detecting deficiencies or disorders of hematopoietic cells. A polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differen- tiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders 35 associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common 40 variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion defi- ciency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or 45 hemoglobinuria. Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or throm- bolytic activity (clot formation). For example, by increasing 50 hemostatic or thrombolytic activity, a polynucleotides or polypeptides, or agonists or antagonists of the present inven- tion could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disor- ders (e.g. thrombocytopenia), or wounds resulting from 55 trauma, surgery, or other causes. Alternatively, a polynucle- otides or polypeptides, or agonists or antagonists of the present invention that can decrease hemostatic or throm- bolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment of heart 60 attacks (infarction), strokes, or scarring. A polynucleotides or polypeptides, or agonists or antago- nists of the present invention may also be useful in treating or detecting autoimmune disorders. Many autoimmune dis- orders result from inappropriate recognition of self as for- 65 eign material by immune cells. This inappropriate recogni- tion results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a poly- 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 97 nucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particu- larly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoim- mune disorders. Examples of autoimmune disorders that can be treated or detected by the present invention include, but are not limited 98 existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent. Examples of hyperproliferative disorders that can be treated or detected by a polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endo- to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye dis- ease. 10 crine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital. Similarly, other hyperproliferative disorders can also be Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polynucleotides or polypeptides, or agonists or antagonists of the present inven- tion. tvloreover, these 11101ecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. 15 treated or detected by a polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary 20 Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above. 25 A polynucleotides or polypeptides, or agonists or antago- nists of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host inmmune cell destruction of the transplanted tissue through an immune 30 response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a poly- nucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particu- 35 larly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to 40 modulate inflammation. For example, the polypeptide or polynucleotide may inhibit the proliferation and differentia- tion of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation asso- 45 ciated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia- reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory 50 bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-l.) Hyperproliferative Disorders A polynucleotides or polypeptides, or agonists or antago- nists of the invention can be used to treat or detect hyper-55 proliferative disorders, including neoplasms. A polynucle- otides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disor- der through direct or indirect interactions. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists 60 of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder. For example, by increasing an immune response, particu- larly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing 65 T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an Cardiovascular Disorders Polynucleotides or polypeptides, or agonists or antago- nists of the invention may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia. Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortop- ulmonary septal defect, endocardial cushion defects, Lutem- bacher's Syndrome, trilogy of Fallot, ventricular heart septal defects. Cardiovascular disorders also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, con- gestive heart failure, congestive cardiomyopathy, paroxys- mal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricu- lar hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmo- nary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis. Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson- White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idio- ventricular rhythm, atrioventricular nodal reentry 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 99 tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycar- dia. Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pul- monary valve insufficiency, pulmonary valve stenosis, tri- cuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis. Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis. Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning. Cardiovascular diseases also include vascular diseases such as aneurYSll1S, angiodysplasia, angioll1atosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay- Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Ler- iche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno- occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno- occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, heredi- tary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency. Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms. Arterial occlusive diseases include arteriosclerosis, inter- mittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans. Cerebrovascular disorders include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and verte- brobasilar insufficiency. Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembo- lisms. Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis. 100 limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis. Polynucleotides or polypeptides, or agonists or antago- nists of the invention, are especially effective for the treat- ment of critical limb ischemia and coronary disease. As shown in the Examples, administration of polynucleotides 10 and polypeptides of the invention to an experimentally induced ischemia rabbit hindlimb may restore blood pres- sure ratio, blood flow, angiographic score, and capillary density. Polypeptides may be administered using any method 15 known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or supposi- 20 torial solid pharmaceutical formulations, decanting or topi- cal applications during surgery, aerosol delivery. Such meth- ods are known in the art. Polypeptides of the invention may be adnlinistered as part of a pharnlaceutical COll1position, described in more detail below. Methods of delivering 25 polynucleotides of the invention are described in more detail herein. Anti-Angiogenesis Activity The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which 30 inhibitory influences predominate. Rastinejad et aI., Cell 56:345-355 (1989). In those rare instances in which neovas- cularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogen- 35 esis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regu- latory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and 40 non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et aI., Biotech. 9:630-634 (1991); Folkman et al.,N. Eng!. 1. Med., 45 333:1757-1763 (1995); Auerbach et aI., 1. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. 1. Opthalmol. 94:715-743 (1982); and Folkman et aI., Science 50 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumu- lated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Sci- 55 ence 235:442447 (1987). The present invention provides for treatment of diseases or disorders associated with neovascularization by admin- istration of the polynucleotides or polypeptides, or agonists or antagonists of the invention. Malignant and metastatic 60 conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art Ischemia includes cerebral ischemia, ischemic colitis, 65 compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral (for a review of such disorders, see Fishman et aI., Medicine, 2d Ed., 1. B. Lippincott Co., Philadelphia (1985)). Ocular disorders associated with neovascularization which can be treated with the polynucleotides or polypep- 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 101 tides or agonists or antagonists of the invention include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with chor- oidal or iris neovascularization. See, e.g., reviews by Walt- man et aI., Am. J. Ophthal. 85:704-710 (1978) and Gartner et aI., Surv. Ophthal. 22:291-312 (1978). Additionally, dis- orders which can be treated with the polynucleotides and polypeptides of the present invention (including agonist and/or antagonists) include, but are not limited to, hemangioma, arthritis, psoriasis, angiofihroma, atheroscle- rotic plaques, delayed wound healing, granulations, hemo- philic joints, hypertrophic scars, nonunion fractures, Osler- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions. Moreover, disorders and/or states, which can be treated with be treated with polynucleotides or polypeptides or agonists or antagonists of the present invention, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibroll1as, trachOll1as, and pyogenic granuloll1as, rheu- matoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis. Diseases at the Cellular Level Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated or detected by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone- dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomeru- lonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. In preferred embodiments, the poly- nucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above. Additional diseases or conditions associated with increased cell survival that could be treated or detected by 102 the polynucleotides or polypeptides, or agonists or antago- nists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lym- 10 phomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, 15 chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, 20 prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, seba- ceous gland carcinoma, papillary carcinoma, papillary adenocarcinoll1as, cystadenocarcinoll1a, ll1edullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, 25 hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, 30 ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Diseases associated with increased apoptosis that could be treated or detected by the polynucleotides or polypeptides, 35 and/or agonists or antagonists of the invention, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders 40 (such as, multiple sclerosis, Sjogren's syndrome, Hashimo- to's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic 45 anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that 50 caused by alcohol), septic shock, cachexia and anorexia. Wound Healing and Epithelial Cell Proliferation In accordance with yet a further aspect of the present invention, there is provided a process for utilizing the polynucleotides or polypeptides, and/or agonists or antago- 55 nists of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal kerati- nocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or 60 antagonists of the invention, may be clinically useful in stimulating wound healing including surgical wounds, exci- sional wounds, deep wounds involving damage of the der- mis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus 65 ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 103 deficiencies and complications associted with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestablishment subsequent to der- mal loss The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are a non-exhaustive list of grafts that polynucleotides or polypeptides, agonists or antagonists of the invention, could be used to increase adherence to a wound bed: auto grafts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterolo- gous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to prOll10te skin strength and to improve the appearance of aged skin. It is believed that the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intesting, and large intestine. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epi- thelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, che- motherapy treatments or viral infections. The polynucle- otides or polypeptides, and/or agonists or antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa. The polynucleotides or polypeptides, and/ or agonists or antagonists of the invention, may also stimu- late healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters 104 nucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, is expected to have a significant effect on the production of mucus throughout the gas- trointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or fol- 10 lowing surgery. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat diseases associate with the under expression of the polynucleotides of the invention. Moreover, the polynucleotides or polypeptides, and/or 15 agonists or antagonists of the invention, could be used to prevent and heal damage to the lungs due to various patho- logical states. A growth factor such as the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, which could stimulate proliferation and differen- 20 tiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting fron1 smoke inhalation and burns, that cause necrosis of the 25 bronchiolar epithelium and alveoli could be effectively treated using the polynucleotides or polypeptides, and/or agonists or antagonists of the invention. Also, the polynucle- otides or polypeptides, and/or agonists or antagonists of the invention, could be used to stimulate the proliferation of and 30 differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmo- nary displasia, in premature infants. The polynucleotides or polypeptides, and/or agonists or 35 antagonists of the invention, could stimulate the prolifera- tion and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., 40 acetaminophen, carbon tetraholoride and other hepatotoxins known in the art). In addition, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with 45 newly diagnosed Types I and II diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, the 50 polynucleotides or polypeptides, and/or agonists or antago- nists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function. Infectious Disease A polypeptide or polynucleotide and/or agonist or antago- 55 nist of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and dif- ferentiation of Band/or T cells, infectious diseases may be treated. The immune response may be increased by either by accelerating reepithelialization of these lesions. The polynucleotides or polypeptides, and/or agonists or antago- nists of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis, are 65 diseases which result in destruction of the mucosal surface 60 enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or poly- nucleotide and/or agonist or antagonist of the present inven- tion may also directly inhibit the infectious agent, without necessarily eliciting an immune response. Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the small or large intestine, respectively. Thus, the poly- 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 105 106 cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis. A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat or detect any of these symptoms or diseases. Preferably, treatment using a polypeptide or polynucle- of the present invention. Examples of viruses, include, but are not limited to the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Her- pes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orth- omyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited 10 otide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or to: arthritis, bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportu- nistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox , hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, 20 leukemia, Rubella, sexually transmitted diseases, skin dis- eases (e.g., Kaposi's, warts), and viremia. A polypeptide or polynucleotide, and/or agonist or antagonist of the present invention can be used to treat or detect any of these symptoms or diseases. 15 polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease. Regeneration A polynucleotide or polypeptide and/or agonist or antago- nist of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist 25 incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage. of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacterial fami- 30 lies and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, 35 Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g., 40 Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococ- cal. These bacterial or fungal families can cause the follow- ing diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, 45 tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis- related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid 50 Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, 55 dermatocycoses), toxemia, urinary tract infections, wound infections. A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat or detect any of these symptoms or diseases. Moreover, parasitic agents causing disease or symptoms 60 that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, 65 Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites can Tissues that could be regenerated using the present inven- tion include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angio- genesis. Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regen- eration of tissues difficult to heal. For example, increased tendon!1igament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide and/or ago- nist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds. Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differ- entiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disor- ders (e.g., spinal cord disorders, head trauma, cerebrovas- cular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Hunting- ton's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Chemotaxis A polynucleotide or polypeptide and/or agonist or antago- nist of the present invention may have chemotaxis activity. 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 107 A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particu- 1ar site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality. A polynucleotide or polypeptide and/or agonist or antago- nist of the present invention may increase chemotaxic activ- ity of particular cells. These chemotactic molecules can then 108 a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate. Additionally, the receptor to which a polypeptide of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et aI., Current Protocols be used to treat inflammation, infection, hyperproliferative 10 disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can he used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be 15 used to treat wounds. in Immun., 1(2), Chapter 5, (1991)). For example, expres- sion cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are It is also contemplated that a polynucleotide or polypep- tide and/or agonist or antagonist of the present invention may inhibit chemotactic activity. These molecules could also grown on glass slides are exposed to the polypeptide of the present invention, after they have been labelled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. be used to treat disorders. Thus, a polynucleotide or 20 polypeptide and/or agonist or antagonist of the present invention could be used as an inhibitor of chemotaxis. Binding Activity Following fixation and incubation, the slides are subjected to auto-lradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an 25 iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor. A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such mol- ecules include antibodies, oligonucleotides, proteins (e.g., receptors),or small molecules. Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Co ligan et aI., Current Protocols in Immu- nology 1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques. Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the mol- ecule. The assay may simply test binding of a candidate com- pound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candi- date compound results in a signal generated by binding to the polypeptide. Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard. Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using As an alternative approach for receptor identification, the labeled polypeptides can be photo affinity linked with cell membrane or extract preparations that express the receptor 30 molecule. Cross-linked material is resolved by PAGE analy- sis and exposed to X-ray film. The labeled complex con- taining the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from 35 microsequencing would be used to design a set of degener- ate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors. Moreover, the techniques of gene-shufiling, motif- shu filing, exon-shufiling, and/or codon-shufiling 40 (collectively referred to as "DNA shu filing") may be employed to modulate the activities of polypeptides of the invention thereby effectively generating agonists and antagonists of polypeptides of the invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 45 and 5,837,458, and Patten, P. A., et aI., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotech- nol. 16(2):76-82 (1998); Hansson, L. 0., et aI., 1. Mol. BioI. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of these patents 50 and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corre- sponding polypeptides of the invention may be achieved by DNA shufiling. DNA shufiling involves the assembly of two or more DNA segments into a desired polynucleotide 55 sequence of the invention molecule by homologous, or site-specific, recombination. In another embodiment, poly- nucleotides and corresponding polypeptides of the invention may be alterred by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other 60 methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptides of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous 65 molecules. In preferred embodiments, the heterologous mol- ecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor 12/22/2014, EAST Version: 3.0.1.1 US 6,632,920 Bl 109 such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphoge- netic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF- betaS, and glial-derived neurotrophic factor (GDNF). 110 directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence. Additional embodiments of the invention are directed to polynucleotides encoding polypep- tides which comprise, or altematively consist of, any com- bination or all of contained in the polypeptide sequences of the invention. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or Other preferred fragments are biologically active frag- ments of the polypeptides of the invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activ- ity. 10 alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta 15 pleated sheet regions in one of the polypeptide sequences of the invention. Additionally, this invention provides a method of screen- ing compounds to identify those which modulate the action Drug Screening Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the com- pound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would nornlally prolif- erate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorpo- ration of 3[H] thymidine. Both agonist and antagonist com- pounds may be identified by this procedure. 20 polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected COll1pound(s) suspected of having antagonist or agonist activity, and assaying the 25 activity of these polypeptides following binding. This invention is particularly useful for screening thera- peutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment 30 employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably trans- formed with recombinant nucleic acids expressing the In another method, a mammalian cell or membrane prepa- ration expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Altematively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and 40 the ability of the compound to bind to the receptor and elicit 35 polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide 45 hydrolysis. All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by 50 activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues. Therefore, the inven- tion includes a method of identifying compounds which bind 55 to the polypeptides of the invention comprising the steps of: (a) incubating a candidate binding compound with the polypeptide; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating 60 a candidate compound with the polypeptide, (b) assaying a biological activity, and (b) determining if a biological activ- ity of the polypeptide has been altered. Also, one could identify molecules bind a polypeptide of the invention experimentally by using the beta-pleated sheet 65 regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are present invention. Thus, the present invention provides methods of screen- ing for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypep- tide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention. Another technique for drug screening provides high throughput screening for compounds having suitable bind- ing affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. 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E-mail: Edzard.Ernst@pms.ac.uk Complementary/alternative medicine (CAM) is a hugely popular subject. Hardly a day goes by that the popular press does not report about one aspect of CAM or another. Currently there are approxi- mately 41 million (!) web sites bombarding the public with information on this topic. Numerous studies have shown how unreliable, and indeed dangerously misleading, this information often is.1,2 To make matters worse, patients cannot rely either on conventional healthcare professionals, who tend to be ill-informed about this subject,3 or on CAM practitioners, who often over-estimate the value of their treatments.4 They cannot even trust the UK ‘official’ patient guide5 (sponsored by the Department of Health), which is promotional, un- informative and inaccurate.6 It is hardly surpris- ing, therefore, that despite the plethora of information, 77% of patients feel ‘insufficiently in- formed about CAM’.7 In this article, I will try to outline how the public is being misled about CAM. Natural equals safe This misunderstanding is as widespread as it is dan- gerous. There is, of course, nothing natural about sticking needles into patients’ bodies (as in acupunc- ture), nor in diluting substances ad infinitum such that the medicines contain nothing but dilutant (as in homeopathy). The fact that in the UK ‘The Comp- lementary and Natural Healthcare Council’ recently started regulating many CAM practitioners8 is therefore misleading in the extreme. Even if a treat- ment is based on what nature supplies (as in herbal medicine), we have to consider its risks seriously. Natural substances may have beneficial health ef- fects but they also can cause harm. For instance, many herbal remedies have the potential to interact with prescribed drugs.9 The risks of CAM are prob- ably less serious than those of synthetic drugs, but they are by no means non-existent or negligible.10 The implication that CAM is natural and therefore safe continues to be used as a powerful tool for misleading the public. CAM defies scientific investigation Enthusiasts of CAM frequently claim that the scien- tific method is not applicable to their field.11 Several reasons are offered for this notion, including: + That the effects of CAM are too subtle to be quantified; + That treatments need to be tailored to each individual and therefore cannot be submitted to testing in clinical trials; + That the therapeutic approach is holistic, which means it cannot be evaluated with reductionistic science. On closer inspection, these arguments turn out to be ill-conceived. They either represent deliber- ate attempts to deceive or they are based on pro- found misunderstandings of what science in general and the clinical trial in particular can achieve. Rather than addressing these issues in detail, it may suffice to point out that even CAM enthusiasts accept scientific investigations of their intervention – as long as their results are positive.12 Yet a lay person may find it difficult to understand methodological issues and thus be easily per- suaded by the claim that CAM defies science. There is no evidence Opponents of CAM sometimes negate the exist- ence of any sound evidence in support of any type of CAM.13 This is clearly wrong. In fact, there are now thousands of clinical trials and in excess of 500 systematic reviews on the subject, many of which suggest that some CAM interventions do generate more good than harm.10 On the other hand, propo- nents of the more exotic forms of CAM – such as Bach Flower Remedies or iridology – often claim that their treatment has not been scientifically evaluated; therefore nobody can say with any degree of certainty that it is not useful. This claim is also wrong. If we only bother to look closely, we do usually find at least some scientific evidence.10 The DECLARATIONS Competing interests None declared Funding None Ethical approval Not applicable Guarantor EE Contributorship EE is the sole contributor Acknowledgements None ESSAY J R Soc Med 2008: 101: 528–530. DOI 10.1258/jrsm.2008.080233528 at US PATENT & TRADEMARK OFFICE on December 22, 2014jrs.sagepub.comDownloaded from reason why enthusiasts negate its existence, I fear, is that it does not confirm their beliefs. Patients can therefore get confused by vociferous and mislead- ing claims about the evidence. CAM saves money If healthcare decision-makers could be convinced today that this notion were true, we would prob- ably have CAM on the NHS tomorrow. Therefore many CAM enthusiasts try to persuade us that integration of CAM would be financially attrac- tive.14 Rigorous evaluations of the reliable evi- dence, however, show this belief to be little more than wishful thinking. On the contrary, the few data available to date suggest that CAM usually constitutes an additional expense, over and above other healthcare costs.10,15 The ‘establishment’ want to suppress CAM Proponents of CAM often claim that their treat- ments are victims of a well-coordinated attack by ‘big pharma’ and ‘the scientific establishment’.16 They seem to believe that these interest groups are secretly plotting to prevent patients from benefit- ing from effective treatments. This notion assumes that the medical profession is a malign clique who would withhold effective treatments from suffer- ing patients for the sole reason that they were developed or discovered by people who do not belong to their profession. Despite the popularity of this argument, there is no evidence that it is true. On the contrary, there is plenty of evidence to show that doctors are more than willing to adopt any treatment that helps their patients, and that ‘big pharma’ takes little – if any – notice of CAM.17 Anecdotes top evidence This widespread feeling was recently put suc- cinctly by Jeanette Winterson: ‘Above all, we should be careful of dismissing the testimony of millions who say the [homeopathic] remedies have worked for them.’11 It is, of course, tempting to assume a causal relationship where only a temporal link exists be- tween an intervention and a clinical outcome. Throughout history, medical progress has been hindered by this misapprehension. But medicine has moved on, and we now know (or should know) that millions can be entirely wrong when assuming that this or that treatment ‘has worked for them’. Clinical improvement can be due to a host of factors other than the specific effect of a treatment,18 yet the anecdote remains perhaps the most effective method by which the press and oth- ers mislead the public. Conclusions Consumers, patients and healthcare decision- makers are regularly being misinformed about CAM. Patients are frequently desperate and therefore vulnerable to such misinformation. Wrong therapeutic decisions are therefore likely. It is our responsibility to see that they receive factu- ally correct information on CAM so that exploita- tion can be avoided and rational healthcare may be facilitated. References 1 Schmidt K, Ernst E. Assessing websites on complementary and alternative medicine for cancer. Ann Oncol 2004;15:733–42 2 Weeks L, Verhoef M, Scott C. Presenting the alternative: cancer and complementary and alternative medicine in the Canadian print media. Support Care Cancer 2007;15:931–8 3 Brown J, Cooper E, Frankton L, et al. Complementary and alternative therapies: survey of knowledge and attitudes of health professionals at a tertiary pediatric/women’s care facility. Complement Ther Clin Pract 2007;13:194–200 4 Schmidt K, Ernst E. Are asthma sufferers at risk when consulting chiropractors over the Internet? Respiratory Med 2003;97:104–5 5 The Prince of Wales’s Foundation for Integrated Health. Complementary Healthcare: a guide for patients. London: FIH; 2005. www.fihealth.org.uk (accessed 08/01/08) 6 Ernst E. Consumer guides for complementary medicine. AIDS & Hepatitis Digest 2005;108:5–6 7 Joos S, Rosemann T, Szecsenyi J, Hahn EG, Willich SN, Brinkhaus B. Use of complementary and alternative medicine in Germany – a survey of patients with inflammatory bowel disease. BMC Complement Altern Med 2006;6:19 8 Hawkes N. New laws to govern alternative medicine. Times Online. 2008 January 5; http://www.timesonline.co. uk/tol/life_and_style/health/article3134337.ece (accessed 08/01/08) 9 Cassileth BR, Lucarelli CD. Herb-Drug Interactions in Oncology. Hamilton, Ontario: BC Decker Inc; 2003 10 Ernst E, Pittler MH, Wider B, Boddy K. The Desktop Guide to Complementary and Alternative Medicine. 2nd edition. Edinburgh: Elsevier Mosby; 2006 11 Winterson J. In defence of homeopathy. Guardian. 2007 November 13; http://www.guardian.co.uk/ print/0,,331242298–103680,00.html (accessed 16/11/07) 12 Mathie RT. The research evidence base for homeopathy: a fresh assessment of the literature. Homeopathy 2003;92:80–7 13 Toynbee T. Quackery and superstition – available soon on the NHS. Guardian Unlimited. 2008 January 8; http://politics.guardian.co.uk/columnist/story/ 0,,2236977,00.html (accessed 08/01/08) 14 Smallwood C. The Role of Complementary and Alternative Medicine in the NHS. An investigation into the potential How the public is being misled about complementary/alternative medicine J R Soc Med 2008: 101: 528–530. DOI 10.1258/jrsm.2008.080233 529 at US PATENT & TRADEMARK OFFICE on December 22, 2014jrs.sagepub.comDownloaded from contribution of mainstream complementary therapies to healthcare in the UK. London: Clarence House; 2005 15 Canter PH, Thompson Coon J, Ernst E. Cost effectiveness of complementary treatments in the United Kingdom: systematic review. BMJ 2005;331:881 16 Ernst E. Why there will never be an alternative cancer cure. Anticancer Drugs 2006;17:1023–4 17 Thompson Coon J, Pittler M, Ernst E. Herb-drug interactions: survey of leading pharmaceutical/herbal companies. Arch Intern Med 2003;163:1371 18 Ernst E. The ‘dirty tricks’ experience can play on us. Postgrad Med J 2007;83:287–8 N E W f ro m R SM P re ss NEW Medico-Legal Title For more information, please visit www.rsmpress.co.uk FRAUD and MISCONDUCT in BIOMEDICAL RESEARCH Fourth edition Edited by Frank Wells and Michael Farthing Fraud and Misconduct in Biomedical Research, now in its fourth edition, boasts an impressive list of con- tributors from around the globe. This publication is an essential textbook on all areas of research fraud and misconduct. £45.00 - 978-1-85315-786-8 - 300 pp - Pbk - September 2008 Journal of the Royal Society of Medicine J R Soc Med 2008: 101: 528–530. DOI 10.1258/jrsm.2008.080233530 at US PATENT & TRADEMARK OFFICE on December 22, 2014jrs.sagepub.comDownloaded from © 2003 Nature Publishing Group P E R S P E C T I V E S to an insoluble resin, purification at each chemical step was simplified to mere rinsing, whereas the optimization of residue addition was facilitated by the use of excess reagents to increase yields. Following the development of automated peptide synthesizers, virtually any sequence could be produced in large quan- tities. The developments of parallel, and eventually split-and-pool, synthesis of pep- tides, as well as the application of this tech- nology to nucleic-acid chemistry2, enabled scientists to generate a much greater diver- sity of linear biopolymers and to keep time and labour to a minimum. Although some successes with these materials as clinical agents have been recorded, orally active, low- molecular-mass small molecules continue to be the preferred choice for drug molecules. In the interest of availing itself of greater collec- tions of such compounds to test in assays, the pharmaceutical industry developed combina- torial synthesis approaches in which drug-like compounds with various rings and functional groups could be assembled in a rapid sequence. Since the first small-molecule com- binatorial library based on a known drug scaffold was introduced in the early 1990s (REF. 3), thousands of unique libraries have been produced, mostly by companies that came to specialize in the business of produc- ing combinatorial libraries, such as Pharma- copeia. In 1996, the CEO of Pharmacopeia, Joseph Mollica, predicted that with the new technology, chemists would boost productiv- ity from tens of novel compounds to nearly 100,000 per year at a fraction of the original cost4. Since 1992, more than 1,250 combina- torial libraries have been described from both academic and industrial laboratories5. As the ability to generate compounds en masse with combinatorial methods became popular, the initial approach taken by most was to play a numbers game. Enormous investments were made by both large and small drug companies in order to purchase the specialized laboratory equipment for combi- natorial synthesis. The initial, sizeable libraries of compounds were often generated as com- pound mixtures. These were then subjected to screens for biological activity with the hope that hits would be rapidly identified. These libraries provided researchers with millions of potential drugs, but their impact was limited by a frequent failure to identify active single entities following the de-convolution of mix- tures exhibiting positive activity.A remedy was attempted by a shift towards the combinator- ial synthesis of pure compounds, but this approach faced formidable challenges inherent to the chemistry involved. Natural products, such as alkaloids, macrolides, and tetracyclines, Laypersons, researchers and clinicians alike speak of the biotechnology revolution with excitement. Media coverage of new breakthroughs in medicine often have the public and the investment community on the edge of their seats, eager for the next blockbuster drug to cure everything from high cholesterol levels to cancer. In this perspective, we examine some of the more popularized and influential new technologies in drug discovery and assess their relative impact on the actual attainment of new therapeutics. The past few years have seen the advent of numerous new technologies and paradigms for drug discovery (TIMELINE). Along with the emergence of each novel method came a promise to generate better drugs in greater numbers while containing or even reducing costs. These techniques have been pursued at least to some extent at most pharmaceuti- cal companies. During the same timeframe, industrial management has come under great pressure from shareholders and other investors to increase the productivity of drug discovery and development to meet annual growth expectations. For many dis- eases, the most obvious approaches to cures have been tried and have often failed. The challenge now is for scientists to attack major diseases with fresh ingenuity. The broader swathes of intellectual property coverage around lead structures, as well as revenue losses from marketed drugs coming off patent, accentuate this demand. In order to obtain a competitive advantage, compa- nies have invested in the newer discovery platforms. A few successes have indeed been registered for each of these technologies, but unrealistic early assessments on the impact of these innovations put forth by the finan- cial community set the stage for sector-wide declines in life science equity valuations. Senior management and scientists in the industry might have also been overly optimistic, as few would now argue that the fruit of these newer efforts have met their initial expectations. Power in (combinatorial) numbers Pharmaceutical companies have traditionally relied on nature’s bounty in their search for new medicinal agents.After selecting an agent from the archives of natural sources, for example the Penicillium mould from which penicillin was derived, scientists would embark on a time-consuming and often frustrating search for what Ehrlich called a “magic bullet”. Positive activity, which could be represented by the ability to kill microbes or cancer cells for instance, would then be followed through serial purification of the crude extracts to give narrower and narrower isolates until the active principle was obtained. Once isolated, the promising leads were usually structurally com- plex, only available in small quantities, and expensive to purify and formulate into medica- ments. These leads, usually against unknown targets and with unspecified mechanisms of action, were handed over to a team of medici- nal chemists who worked to make simpler, more bioavailable compounds based on the core structure of the lead compound. Medicinal chemistry is often viewed as a limiting factor in the creation of new drugs, as the steps involved in the preparation of just a single compound are labour-intensive. The bench chemist carrying out synthesis and purification could only be expected to pro- duce a certain number of new compounds per year. For biochemists working with pro- teins, the widespread adoption of Merrifield’s method for the linear solid-phase synthesis of peptides profoundly altered the nature of their work1. By attaching the nascent peptides NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | APRIL 2003 | 321 A brief history of novel drug discovery technologies Leland J. Gershell and Joshua H. Atkins T I M E L I N E “For many diseases, the most obvious approaches to cures have been tried and have often failed. The challenge now is for scientists to attack major diseases with fresh ingenuity.” © 2003 Nature Publishing Group 322 | APRIL 2003 | VOLUME 2 www.nature.com/reviews/drugdisc P E R S P E C T I V E S applying the new techniques of molecular biology to basic problems in medicine. They pioneered the novel area of protein-based drugs, and developed highly successful busi- ness models on the simple scientific notion that replacing a protein deficient in a disease state would be of clinical benefit. The early suc- cesses of drugs such as Epogen and recombi- nant human insulin provided a tremendous stimulus to the biotech industry and lent strong support to the notion that drugs need not be small molecules, and could be made by cells in vats on a large scale without requiring tedious manipulation by chemists at the bench. The success of the early protein-based drugs, combined with new developments in cancer biology as partially stimulated by an increased understanding of gene expression, led to the monoclonal antibody ‘revolution’. The Genentech/IDEC launch of Rituxan in 1997 for non-Hodgkin’s lymphoma left the biotech world and its investment community awe struck by the seemingly limitless potential of antibodies. The scientific basis of this idea was simple — proteins expressed specifically in certain diseases that were displayed on the surface of cells, or which circulated in the bloodstream, could be targeted by antibodies directed against them. These antibodies would act like a precision defence system to either neutralize the toxic molecules, or specifically deliver medications only to those tissues that bore them. generated information about biological targets that has enabled chemists to prepare libraries geared towards selected target classes8. Furthermore, a combinatorial approach to generating sub-libraries, in which pendant groups are selectively fixed or varied around a common core, might provide a multivariable dataset that can be evaluated during the screening process to reveal interdependent relationships of structural variations; this information can then guide subsequent hit- to-lead optimization steps9. This distinction is usually absent from individual experiments that examine only one parameter at a time. In this way, the mode of utility of combinatorial chemistry has shifted from ‘lead discovery’ to ‘lead optimization’. A few companies are finding this approach productive, and now have selected compounds in clinical trials. It can be safely said that the overall quality of combi-chem libraries has improved appre- ciably, increasing the odds that hopes for new leads will be met with satisfaction. The allure of the ‘omics’ While the revolutions in chemistry were taking hold, advances in molecular and cell biology were also attracting attention in the pharma- ceutical industry. In the late 1970s and early 1980s, recombinant DNA technology and the human genome were buzzwords, and compa- nies such as Genentech and Amgen led the way in the nascent biotechnology market by which have traditionally been a fertile source of pharmaceutical leads, are characterized by complex, highly functionalized, polycyclic sys- tems bearing multiple stereocenters. Their preparation by standard organic synthesis can require more than 20 chemical operations with almost as many purifications, which pre- sents a daunting and inefficient task for the bench chemist. As chemists were eager to take advantage of the low-hanging fruit by making libraries guided by simpler chemistries, it is not surprising that the early libraries were composed of compounds that were not neces- sarily ‘drug-like’ in nature. Although the past two decades have seen huge advancements in methods for stereo- and enantioselective syn- thesis in solution, the adaptation of these methods to solid-phase chemistry has been slow, but is gradually taking hold6.At the same time, sophisticated techniques for stereoselec- tive solid-phase oligosaccharide synthesis, such as those undergoing refinement at Optimer, bode well for the generation of thera- peutics based on this biologically important class of molecules7. Chemical hurdles aside, a number of com- panies that tried the ‘massive library’ model soon abandoned it, often to re-orient their efforts toward generating smaller, focused libraries with permutations around a handful of promising scaffolds. These efforts have been aided by a better understanding of chemical versus biological structure spaces, which has Protein Data Bank of X-ray crystal structures established with 12 structures. Kohler and Milstein report first monoclonal antibodies. Bruce Merrifield awarded Nobel Prize for solid-phase peptide synthesis. FDA approval for human insulin (Genetech/Lilly). OKT3 approved as the first non-human monoclonal antibody therapeutic. Pharmacopeia founded in Princeton, NJ. Affymetrix, the first microarray/microchip company, founded. Aptamers discovered. Vitravene (Isis/Novartis) becomes the first antisense drug approved for treatment of cytomegalovirus retinitis. Cancer, cardiovascular disease and stroke remain leading causes of death in the United States. McKinsey/Lehman ‘Fruits of Genomics’ report predicts productivity crisis for pharmaceutical industry. 1971 1975 1982 1984 1986 1989 1990 1991 1992 1993 1997 1998 2000 2001 2002 2003 FDA approval for Epogen (Genetech). Draft sequence of the human genome published in Nature and Science. Big pharma R&D budget averages US $800 million per new chemical entity. Ellman reports combinatorial benzodiazepine library. First gene therapy experiment in humans. Human Genome Project initiated. FDA approval for Rituxan (Genentech/IDEC). Venture capital funding for biotechnology ventures peaks at US $6 billion. The first protein-based therapeutic First transgenic or ‘knockout’ mouse. The first humanized monoclonal antibody for cancerViracept. An HIV protease inhibitor, approved by FDA. FDA approves 17 NCEs, compared with 23 in 1990; approves 44 new formulations of old drugs. Lipitor (Pfizer) annual sales reach US $8 billion. More than 2,500 companies worldwide engaged in the application of novel technologies to drug discovery. Timeline | Selected evolutionary landmarks in drug discovery © 2003 Nature Publishing Group P E R S P E C T I V E S method has been hindered in the past by a number of factors, including the time it takes to produce and study knockout or transgenic animals, as well as the possibility that com- pensatory mechanisms might alter the pheno- type. Companies specializing in the transgene field have begun to approach the technology more aggressively. For example, scientists at Lexicon Genetics have identified 5,000 targets for which they plan to generate knockout The principal technological advancement that permitted the use of antibodies as thera- peutics related not to genomics, proteomics or combinatorial chemistry, but to mono- clonal technology that enabled scientists to ‘humanize’ mouse or other animal antibody proteins so that they would not be recognized as foreign by the immune system10. Mono- clonal antibodies had been produced and characterized since 1975 (REF. 11), and shortly after were in development as therapies to pre- vent transplant rejection. However, with the advent of the new humanization technology and its subtle variations, companies such as Abgenix, Medarex and Protein Design Labs took the biotech community by storm, with pharmaceutical partnerships and new prospects being announced on a frequent basis. In fact, in 2000 nearly 25% of all new drugs in development were believed to be monoclonal antibodies, with 90 monoclonal antibodies in clinical trials12. In 2003, monoclonals continue to have a high profile, but with the tempered optimism that usually settles on the biomedical commu- nity as the challenges of implementation become clearer. In late 2000, analysts could not stop talking about the upcoming billion-dollar drug Xolair — a monoclonal antibody tar- geted at IgE for asthma, in development by Genetech, Novartis and Tanox. By the summer of 2001, the FDA was requesting additional data, indicating that the potential for patient treatment, at least initially, would be less than that originally projected. Xolair remains in development, which began more than a decade ago, and it is unlikely to see the market before 2004. The Human Genome Project, which was launched in the late 1980s, heralded a revolu- tion in medicine, and provided further promises for new therapeutics. It was the start of a media frenzy, with a New York Times piece nearly 15 years ago suggesting that “in the not-so-distant future, we can expect to walk into a physician’s office for an annual physical and walk out with a blueprint of our genetic inheritance — and with the know- ledge of the most likely cause of our own death”13. As scientists, we have learned to temper the enthusiasm of the media with our own understanding of nature’s devilish com- plexity and redundancy. At the same time, the surge of gene and protein data in the late 1990s led to the irresistible idea that once all of the disease targets were characterized, drugs for each would eventually follow suit. However, as target validation has lagged behind the greater access to potential targets, the chances for failure have actually increased. So, the importance of sufficiently validating of a target before embarking on a drug-discovery project must be underscored, and indeed the requirement for validation has been a driving force for the development of many new techniques in drug discovery. Targeted gene disruption in whole animals, such as knock- outs or transgenic models, has been increas- ingly used over the past decade to determine the relevance of a gene implicated in a partic- ular pathway or phenotype14. However, this NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | APRIL 2003 | 323 Box 1 | Human immunodeficiency virus — new science and new targets As a group, the anti-human immunodeficiency virus (HIV) therapeutics offer a shining example of how the newer drug discovery techniques have borne great commercial success. These drugs have enabled the HIV-infected patient to transform what was essentially a death sentence into a manageable, long-term disease. Although drug resistance by the virus remains an issue, options for combination therapy against multiple viral proteins has further opened the window for intervention. HIV protease was identified in the late 1980s as a potential therapeutic target. It functions in the assembly and maturation of virus particles, and its inactivation was found to lead to noninfectious virions. The crystal structures of the HIV proteases became available shortly after, both in isolation and in complexes with numerous inhibitors. Computational studies of these inhibitor complexes were reported in the early 1990s, and their modes of binding could be investigated with the aid of molecular mechanics. At this time, the small companies Agouron and Vertex had become dedicated to developing drugs against HIV. Both of these companies had been established in the 1980s for the express purpose of creating small-molecule drugs through rational design. Techniques such as protein crystallography, nuclear magnetic resonance and computational biochemistry would be brought to bear on building compounds ‘atom-by-atom’ against protein targets in disease. Nelfinavir (Viracept; Agouron) gained FDA approval in 1997 for the treatment of HIV infection, and generated US $43.6 million in sales in its first quarter on the market. Two years later, amprenavir (Agenerase; Vertex/GlaxoSmithKline) received FDA approval. There are now six protease inhibitors on the market. In 2003, the development of new drugs for HIV infection continues, and efforts are now directed towards additional targets, including HIV-1 integrase33. This enzyme allows HIV DNA to integrate into human cellular DNA. Obtaining a complete X-ray crystal structure of this protein has proved more difficult than for HIV protease, but with partial structures and computer models in hand, the industry is working to apply the principles of rational and structure-based drug design to this new challenge, with novel agents lurking on the horizon. Suggest better targets for intervention Collaborator data Literature data Chemistry data Biology data Computational chemistry software Computational biology software Optimize ADMET bioactivity data Prioritize molecules In vivo Figure 1 | Virtual drug discovery. Computational approaches represent a hopeful means to leverage biological and physico-chemical data to drive the structural optimization of compounds for their interaction with selected targets. Similarly, absorption, distribution, metabolism, excretion and toxicology (ADMET) algorithms could serve to weed out problematic candidates early on. Such screening of ‘virtual compounds’ is likely to be faster and cheaper than wet methods, a boon to an industry suffering rising costs of development. Adapted from Ekins, S., et al.J. Comput. Aid. Mol. Des. 16, 381-401 (2002) © Kluwer Academic Publishers. © 2003 Nature Publishing Group P E R S P E C T I V E S Today, however, the large majority of new drugs are still directed at a very small subset of targets — mostly G-protein-coupled receptors and other enzymes that have been well-characterized for years. Can disease be rationally eradicated? Although companies have been discovering drugs for over a century, it has only been in the past few decades that depictions of pro- tein targets, sometimes with their cognate ligands bound, have become available. As the resolution of this three-dimensional informa- tion has improved greatly, companies have sought to ‘rationally’ design drugs from scratch that would selectively interfere with cellular function. A Washington Post article in 1988 carried the headline ‘Computer is Drug Design’s New Mortar and Pestle’18 and high- lighted new efforts in rational drug design, stimulated by research on drugs to treat infec- tion with the human immunodeficiency virus (HIV). There was much talk of integrating chemists, X-ray crystallographers, cell bio- logists and others into teams that might expeditiously assimilate new data for the design of precisely targeted drugs for any disease in which a causative protein was implicated. Agouron, a small pharmaceutical company based in San Diego, demonstrated the elegance and utility of this approach with the approval of the HIV protease inhibitor nelfinavir (Viracept) in 1997. The drug was designed specifically to bind to a portion of the HIV protease enzyme, which was first characterized in 1987, using computer mod- elling against the X-ray crystal structure of the enzyme (BOX 1). It was a remarkable success and eventually led to the acquisition of Agouron by Pfizer. Unfortunately, outside of HIV protease inhibitors only a few other rationally designed small-molecule drugs have followed — most notably Relenza for the prevention of influenza infection. However, the simple notion that with the right technology and an integrated discov- ery team one could proceed de novo from gene, to protein and its three-dimensional structure, and finally to a drug, created a whirlwind in the pharmaceutical commu- nity. Almost all of the major pharmaceutical companies adopted this strategy to varied extents internally or in partnership. The evolution of faster methods for gen- erating protein structures has paved the way for companies to specialize in the high- throughput structural determination of target proteins with arrays of putative inhibitors. Two biotech ventures with high-profile man- agement and ample capital have come to the fore — Structural Genomix and Syrrx. These models. In the ever-more-automated state of drug discovery technology, it is suspected that target validation will continue to remain a challenge. One of the most realistic promises of the Human Genome Project has in fact been realized — a large increase in the number of potential drug targets. Indeed, one of the broadest changes in drug discovery efforts has been a shift towards an inductive model, in which the molecular pathways and entities believed responsible for a disease — or which at least have a causal role in the disease — form the starting point in the search for new drugs. We can expect that these heretofore unknown targets, following appropriate validation, will generate new perspectives on the treatment of diseases that have been resistant to therapies, such as neurological disorders and obesity. mice in which they can study mechanisms of disease and potential therapeutic candidates15. Moreover, the recent completion of the sequencing of the mouse genome might provide further insights into the functional differences between human and mouse sys- tems, and could eventually enhance the transgenic approach to target validation and lead development. Antisense technology, which offers a means to validate targets by preventing pro- tein expression of a particular mRNA species, has been used with some success16. Newer antisense approaches, including the application of chimeric small nuclear RNA (snRNA) to control gene expression, are par- ticularly promising17. Multigene disruption in single animals is an emerging technology that could augment the data gained from knockout Box 2 | On the cutting edge — pharmacogenomics Nearly half all of drugs fail due to unforeseen toxicity or metabolism issues, and liver toxicity is the most common reason for FDA drug withdrawal or restriction (see table). Improved success in the late stages of drug development might lie in the ability to anticipate side-effects, interactions with other medications, and the structure and bioactivity of drug metabolites. As genetic variability is an important factor in determining the differences in drug responses among people, ‘fingerprints’ of individual human genomes might one day be used to tailor the selection of patients for clinical trials, or to select specific medications for an individual. The technology for assessing individual genetic variations in every aspect of disease, including metabolic processes, might also help drug companies to further study toxic side effects seen in a small minority of patients, thereby allowing the companies to refine dosing guidelines rather than withdrawing drugs from the market. Recently, UK-based Astex Technology reported the X-ray structure of the liver-based cytochrome p450 enzyme CYP3A4, which is believed to be responsible for the metabolism of at least 50% of therapeutics presently used. Structural knowledge of the enzymes involved in metabolism, and how they vary within populations, could inform drug discovery efforts so as to reduce the likelihood of untoward toxicity. Drug Company Disease/indication Toxicity Action Baycol Bayer High cholesterol Rhabdomyolysis Withdrawn levels 2001 Duract Wyeth-Ayerst Pain Liver toxicity Withdrawn 1998 Fen-phen Wyeth-Ayerst Obesity Cardiac arrhythimia Withdrawn 1998 Lotronex GlaxoSmithKline Irritable bowel Ischemic colitis Withdrawn syndrome 2000 Propulsid Janssen Abnormal Cardiac arrhythmia Withdrawn gastrointestinal 2000 motility Raplon Organon Anesthesia Bronchospasm Withdrawn 2001 Rezulin Parke-Davis/ Type II diabetes Liver toxicity Withdrawn Warner-Lambert 2000 Seldane Hoechst Allergy Cardiac arrhythmia Withdrawn 1998 Serzone Bristol-Myers Depression Liver toxicity Selective Squibb withdrawal 2003 Trovan Pfizer Anti-microbial Liver toxicity New restrictions Zyflo Abbott Asthma Liver toxicity New restrictions 324 | APRIL 2003 | VOLUME 2 www.nature.com/reviews/drugdisc © 2003 Nature Publishing Group P E R S P E C T I V E S in terms of what is relevant to the nature of composition patents), are often met with disappointment in activity assays because too small a sample set of resulting com- pounds is tested. In any case, the great number of algorithms and software products avail- able for virtual screening both attest to the hopes for this technology as well as to the fact that the pharmaceutical community has yet to settle on defined standards. Siliconization and automation In parallel with the development of rational drug design, combinatorial chemistry, and a variety of genome and proteome projects, there has been a growing fascination with silicon chips and robotics. This was stimu- lated, in part, by the increasing reliance of drug discovery on computer technology and simulation, but even more so by the need to screen the large number of drugs and targets created by other breakthrough develop- ments. Silicon chips have provided some degree of automation and high-throughput and have also reduced the amounts of test reagents needed, saving both laboratory time and money. Investigations once con- sidered to be unrealistic because of the sheer number of individual experiments that companies are leveraging new technology in rapid, high-throughput protein crystalliza- tion with advances in computer modelling programs, similar to those used at Agouron, to better understand binding modes and then to use this information to design superior ligands. It remains to be seen whether advances in biophysical chemistry are yet robust enough for these predictive technologies to translate generally into successful drug candidates. There already exist substantial databases of both public and proprietary crystal structures of validated clinical targets, and yet few drugs seem to be reaching the market as a result. One case in point is British Biotech’s experience with marimastat, a potent small-molecule inhibitor of matrix metalloproteinases (MMPs), a class of enzymes known to be involved in a range of diseases from arthritis to cancer19. The drug failed numerous clinical trials despite the strong rational basis of its design. Moreover, other companies have continued to explore the fertile ground of MMP inhibitors, but to date, despite more than 15 available crystal structures, large numbers of preclinical and clinical candi- dates and tremendous resources, no drugs aimed toward these enzymes have success- fully reached the market. This experience serves as a lesson that although structure- based design has evolved into a viable method for the discovery of inhibitors, there is no escaping the manifold physiological requirements put on a compound to qualify as a real drug. There is little doubt that rational drug design will remain an impor- tant force in drug development, but success in this effort still requires extensive empirical experimentation and remains subject to the surprises and challenges inherent in all approaches to drug development. Virtual screening The ability to model and predict the binding sites of proteins on the one hand, coupled with large databases of small-molecule com- pounds on the other, has encouraged the development of software that iteratively screens compounds in silico (FIG. 1). These tools have become more integral to the search for new drugs as computing power has increased and become cheaper. The potential of this approach has already been demonstrated by the identification of several inhibitors and antagonists. Although these efforts might serve to accelerate the preclini- cal drug discovery process, their usefulness remains limited by the predictive value of the compound ‘descriptors’, as well as by the differences between molecular structures that can be conceived versus those that can be readily synthesized. Although diversity is crucial, it is generally more desirable to design an ‘informed’ virtual library that con- tains synthesizable and drug-like compounds than it is to generate a library that maximally samples diversity space with billions of ran- dom compounds. This is in agreement with recent studies showing that within the current pharmacopeia, there are only about 10,000 drug-like compounds20, and that the diver- sity of shapes in the set of known drugs is quite low21. Computational teams in phar- maceutical companies often embark on the search for drug candidates for molecular targets that already have established lead compounds; in this case, they are attempting to find structures that are sufficiently differ- ent so as to circumvent outside patents. Although appealing, virtual screens might be less productive than anticipated in this regard because the chances of finding a true lead — that is, a high-potency inhibitor — are far lower than those of simply finding a hit that represents a much earlier point in the optimization process. Moreover, efforts by some to mine compound databases for molecules that show similarity to the finger- print of a given lead (but for which the two- dimensional representation is quite different, NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | APRIL 2003 | 325 Box 3 | Congestive heart failure — still looking for better drugs Mortality rates for congestive heart failure (CHF) remain high, with most estimates reporting a mortality of 50% within five years of diagnosis. Early models of CHF suggested that a reduction in blood flow to the kidneys as a result of poor cardiac output induces compensatory mechanisms that retain salt and water. This yields a volume-overloaded state, with the result that peripheral vasoconstriction to maintain blood pressure becomes excessive. Two of the more commonly prescribed drugs approved to treat CHF — the β-adrenoceptor antagonist carvedilol (Coreg; GlaxoSmithKline) and the angiotensin-converting enzyme inhibitor enalapril (Vasotec; Merck) — were originally approved as antihypertensives. They do happen to antagonize the sympathetic and neurohormonal elements of CHF, however, and have proven beneficial for CHF patients. At the same time, our current understanding of CHF has matured into a complex, multidimensional model in which numerous targets have been identified for therapeutic intervention. Potential new therapeutics include endothelin-receptor antagonists, neutral- endopeptidase inhibitors (NEPIs), angiotensin-receptor antagonists and aldosterone antagonists34. Drugs in some of these categories are already on the market as antihypertensives, including valsartan (Diovan; Novartis), spironolactone (Aldactone; Searle) and bosentan (Tracleer; Actelion/Genentech). Bristol-Myers Squibb recently had the novel drug omapatrilat (Vanlev) in the NEPI class, but it was withdrawn from clinical trials due to unforeseen side effects. However, even if Vanlev had been approved, it had previously been shown that Vanlev was no more effective for CHF than established therapies. Drug manufacturers are now aggressively pursuing label expansions for existing antihypertensives to treat CHF as the potential market opportunities are enormous. Regardless, the fact remains that none of the above medications has been shown to substantially reduce the overall course of CHF in most patients. Unfortunately, heart transplantation remains the only definitive intervention. Adenosine receptors are now CHF targets of current interest at companies such as Aderis Pharmaceuticals and CV Therapeutics, but it remains to be seen whether drugs directed towards the adenosine pathway will have a superior clinical effect. As the link between basic science and clinical reality is often tenuous, many hypotheses to explain CHF have ultimately failed when applied to the quest for better drugs. © 2003 Nature Publishing Group 326 | APRIL 2003 | VOLUME 2 www.nature.com/reviews/drugdisc P E R S P E C T I V E S disproportionate failure of drugs during clinical trials, a great deal of effort has focused on developing better predictive models of how compounds will act in the body. Ensuring that a compound meets ADMET standards can involve hundreds of tests in cell-based assays and animal models, some of which require large quantities of compound. The process represents a significant bottle- neck, as combinatorial chemistry and high- throughput screening make available only small quantities of compounds for screening. One approach that has met with some success has been taken by Deltagen, and involves the use of transgenic mice bred to express specific genes involved in drug toxicity or, in the case of cancer, specific drug-resistance genes28,29. A number of tissue-based systems that use cell lines have been developed for the determina- tion of absorption and distribution properties established (for example, Caco-2 for intestinal absorption), but there is still a dearth of assays for predictive toxicity. Also, data generation tends be relatively slow in comparison with the rate of compounds processed by high- throughput activity screens. This might be the main reason why ADMET profiling is not applied earlier in the drug discovery process, that is, before many compounds have been excluded based on activity alone.A more pro- gressive approach has been the adoption of parallel processing, in which potential drugs are put through ADMET screens at every step of the development process to reduce surprises in the ‘end-game’30. This is particularly applica- ble to the design of combinatorial libraries15. However, this approach demands that the basic parameters of drug metabolism be well- defined, which, generally, they are not, despite Lipinski’s ‘rule-of-five’31,32. The cutting-edge solution to this problem is the emergence of ADMET prediction soft- ware as developed by companies such as ArQule and Schrödinger. Their products are based on predictive models that allow the vir- tual screening of compounds according not only to parameters such as membrane per- meability and lipid solubility, but to the actual chemistry of metabolism of the cytochrome p450 system. Although highlighted by the press as a new drug development technology, these programs are essentially an outgrowth of quantitative structure–activity relationship (QSAR) approaches that have been around for some 30 years and to which the latest computational methods, as well as accumu- lated basic and clinical knowledge, are now being applied. In any case, these promising methods often suffer from poor validation and inaccuracies, although access to larger databases of known compounds and their yet approved that have come directly out of merged efforts between combinatorial chemistry and high-throughput screening9, although inhibitors to thrombin, the cathep- sins, and p38 MAP kinase are presently in clinical trials. The perfect drug that fails in the clinic All too frequently, a compound that works well in the laboratory and in preclinical trials, and even in small patient samples, meets with disaster when applied to larger study groups (BOX 2). In fact, it has been estimated that at least 50% of development candidates fail because of problems with absorption, distri- bution, metabolism, elimination and toxic- ity (ADMET), and about half of drugs on the market suffer some kind of ADMET problem26. Even after many collective years of knowledge accumulated by scientists in the industry, it remains a real challenge to predict these pharmacokinetic properties from just the structure of a compound27. Given the would be involved are now carried out in days, or even hours. The original Affymetrix concept of diagnostic chips for every pathology has yet to be realized, but tech- nologies such as GeneChip arrays have been generously applied, particularly in the area of differential nucleic-acid expression in health and disease22,23. The recent shift of focus to proteins, with the technological challenges inherent in the production of protein-, cell- and tissue-coated silicon chips, as well as in the optimization of sensi- tive and specific detection systems, has slowed the progress of this technology24. There also remain challenges in the area of quality control and correlation to in vivo data. At this stage, silicon technologies are primarily tools for generatin interesting data for early stage screening and mechanism- based studies, but as the technology comes of age there is tremendous potential for applications to every aspect of the develop- ment process25. To date, there are no drugs a b c d e Figure 2 | Emerging technologies and their developers. a | Automated two-dimensional gel electro- phoresis for the high-throughput purification of complex mixtures of proteins at very low concentrations35 (Amersham Biosciences, Applied Biosystems and Millipore). b | Tissue microarrays36, which are microchips coated with diverse types of normal and disease human tissues for the high-throughput validation of targets and the evaluation of efficacy, distribution and toxicity of drugs in the context of native complex biochemical milieu (Beecher Instruments, Biolog, Diomeda Life Sciences and Invitrogen). c | Directed evolution, which involves the engineering of protein/antibody therapeutics and micro- organisms by controlled manipulation of cellular machinery through strategic, directed mutagenesis and chemical gene synthesis37 (Applied Molecular Evolution, Maxygen and Morphotek). d | Aptamer technology. Aptamers are functional nucleic acids that bind specific regions of proteins to modulate their function in vivo, and have applications in target validation, therapeutics and diagnostics38 (Aptanomics, Archemix, Gilead Sciences and Somalogic). e | Predictive and in silico absorption, distribution, metabolism, excretion and toxicity platforms. Simulation software, microarrays, and bioinformatics mining and processing technologies are used to optimize the performance of clinical drug candidates in late- stage clinical trials and beyond27 (Amedis, Arqule, Cyprotex, Schrödinger and Tripos). © 2003 Nature Publishing Group P E R S P E C T I V E S 21. Bemis, G. W. & Murcko, M. A. Properties of known drugs. 1. Molecular frameworks. J. Med. Chem. 39, 2887–2893, (1996). 22. Golub, T. R. et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 286, 531–537 (1999). 23. Scherf, U. et al. A gene expression database for the molecular pharmacology of cancer. Nature Genet. 24, 236–244 (2000). 24. Kodadek, T. Protein microarrays: prospects and problems. Chem. Biol. 8, 105–115 (2001). 25. Gerhold, D. L. et al. Better therapeutics through microarrays. Nature Genet. 32, 547–551 (2002). 26. Hodgson, J. ADMET — turning chemicals into drugs. Nature Biotechnol. 19, 722–726 (2001). 27. Ekins, S. & Rose, J. In silico ADME/Tox: the state of the art. J. Mol. Graph. Model 20, 305–309 (2002). 28. Schmitt, C. A. & Lowe, S. W. Apoptosis and chemoresistance in transgenic animal models. J. Mol. Med. 80, 137–146 (2002). 29. Zhang, J. et al. Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. Science 298, 422–424 (2002). 30. Tarbit, M. H. & Berman, J. High-throughput approaches for evaluating absorption, distribution, metabolism and excretion properties of lead compounds. Curr. Opin. Chem. Biol. 2, 411–416 (1998). 31. Lipinski, C. A. et al. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 23, 3–25 (1997). 32. Beresford, A. P. et al. The emerging importance of predictive ADME simulation in drug discovery. Drug Discov. Today 7, 109–116 (2002). 33. Craigie, R. HIV integrase, a brief overview from chemistry to therapeutics. J. Biol. Chem. 276, 23213–23216 (2001). 34. Klapholz, M. Emerging therapies in heart failure. Cardiol. Spec. Ed. 7, 71–74 (2001). 35. Michels, D. A. et al. Fully automated two-dimensional capillary electrophoresis for high sensitivity protein analysis. Mol. Cell. Proteomics 1, 69–74 (2002). 36. Perkel, J. M. Tissue microarrays: advancing clinical genomics. The Scientist 21, 39 (2002). 37. Cornish, V. & Tao, H. Y. Milestones in directed enzyme evolution. Curr. Opin. Chem. Biol. 6, 858–864 (2002). 38. James, W. Nucleic acid and polypeptide aptamers: a powerful approach to ligand discovery. Curr. Opin. Pharmacol. 1, 540–546 (2001). Acknowledgements The authors wish to acknowledge helpful discussions with the associate consultants and advisors of AGW BioStrategy. Online links DATABASES The following terms in this article are linked online to: Cancer.gov: http://www.cancer.gov/cancer_information/ Non-Hodgkin’s lymphoma LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ P38 MAP kinase Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/Omim/ Asthma | Congestive heart failure FURTHER INFORMATION 5Z.com: http://www.5z.com Bio.com: http://www.bio.com The National health Museum: http://www.accessexcellence.org Access to this interactive links box is free online. properties has allowed the software developers to offer greater reliability in their latest releases32. Overall, the most successful tech- nologies will provide in silico predictive infor- mation that will identify the most effective balance among therapeutic efficacy, potency, bioavailability and toxicity. Conclusions The past two decades have witnessed remark- able progress in our understanding of the genetic and mechanistic basis of disease, the central importance of intercellular communi- cation in the disease process and the potential success of highly targeted therapeutics. There has been both a rapid explosion in the amount of data available to researchers trying to create new drugs, and a parallel rise of new technologies in screening and automation to allow scientists to manage and sort the incoming data. In the course of achieving these goals, new challenges have been identi- fied. In particular, the frequent and highly unpredictable failure of drugs in human trials is increasingly recognized as a roadblock to greater productivity in drug development. The full impact of the numerous genome and proteome projects has yet to be felt with regard to the long-promised development of genotype- or phenotype-specific therapeu- tics, and predictive technologies for drug dis- position in the body remain at a primitive level. Overall, there are indications that pro- ductivity in the pharmaceutical industry is decreasing, that recent technologies have not reduced the cost of drug discovery and that the means for creating data have outpaced the ability to interpret and apply the data. Indeed, to date, the biotech revolution has made only a tangential contribution to the management and treatment of the major diseases facing society, such as congestive heart failure (BOX 3), cancer, asthma and sepsis. As with many developments in medicine, optimistic predictions and hyperbole con- tribute to the development of unreasonable expectations for new therapeutics. Despite substantial technological advancement, how- ever, it remains difficult to imagine a day without the spectre of diseases such as cancer, congestive heart failure and Alzheimer’s dis- ease. A number of newer technological avenues, having already shown some promise, are now being travelled with hopes for success (FIG. 2). Although we might look forward to groundbreaking therapies in the future, as a general rule, experience wins over expectation. AGW BioStrategy, 55 West 26 Street, Suite 10D, New York, New York 10010, USA. Correspondence to L.J.G. e-mail: consultant@agwgroup.com doi:10.1038/nrd1064 1. Merrifield, R. B. Solid-phase syntheses (Nobel lecture). Angew. Chem. 97, 801–812 (1985). 2. Elmblad, A. et al. Synthesis of mixed oligodeoxyribonucleotides following the solid phase method. Nucleic. Acids Res. 10, 3291–3301 (1982). 3. Bunin, B. A. & Ellman, J. A. A general and expedient method for the solid-phase synthesis of 1,4-benzo- diazepine derivatives. J. Am. Chem. Soc. 114, 10997–10998 (1992). 4. Day, K. Testing a combination punch against disease. The Washington Post (Washington) (April 14, 1996). 5. Dolle, R. E. Comprehensive survey of combinatorial library synthesis: 2000. J. Comb. Chem. 3, 477–517 (2001). 6. Arya, P. et al. Toward high-throughput synthesis of complex natural product-like compounds in the genomics and proteomics age. Chem. Biol. 9, 145–156 (2002). 7. Sears, P. & Wong, C.-H. Toward automated synthesis of oligosaccharides and glycoproteins. Science 291, 2344–2350 (2001). 8. Wess, G. How to escape the bottleneck of medicinal chemistry. Drug Discov. Today 7, 533–535 (2002). 9. Auld, D. S. et al. Targeting signal transduction with large scale combinatorial collections. Drug Discov. Today 7, 1206–1213 (2002). 10. Hughes, D. Therapeutic antibodies make a comeback. Drug Discov. Today 3, 439–442 (1998). 11. Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975). 12. To, A. et al. Monoclonal antibodies: present and future. Credit Suisse First Boston Corporation (2000). 13. Schmeck, Jr, H. M. Gene studies emerging as key engine of science. The New York Times (September 6, 1988). 14. Debouck, C. & Metcalf, C. The impact of genomics on drug discovery. Annu. Rev. Pharmacol. Toxicol. 40, 193–208 (2000). 15. Zambrowicz, B. P. & Sands, A. T. Knockouts model the 100 best-selling drugs — will they model the next 100? Nature Rev. Drug. Discov. 2, 38–51 (2003). 16. Schmitt, C. A. & Lowe, S. W. Apoptosis and chemoresistance in transgenic animal models. J. Mol. Med. 80, 137–146 (2002). 17. De Angelis, F. G. et al. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Δ 48–50 DMD cells. Proc. Natl Acad. Sci. USA 99, 9456–9461 (2002). 18. Specter, M. Computer is drug design’s new mortar and pestle. Washington Post (December 17, 1988). 19. Whittaker, M. et al. Design and therapeutic application of matrix metalloproteinase inhibitors. Chem. Rev. 99, 2735–2776 (1999). 20. Lipinski, C. A. Drug-like properties and the causes of poor solubility and poor permeability. J. Pharmacol. Toxicol. Methods 44, 235–249 (2000). NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | APRIL 2003 | 327 Molecular obesity, potency and other addictions in drug discovery Michael M. Hann* Received 18th January 2011, Accepted 14th February 2011 DOI: 10.1039/c1md00017a Despite the increase in global biology and chemistry knowledge the discovery of effective and safe new drugs seems to become harder rather than easier. Some of this challenge is due to increasing demands for safety and novelty, but some of the risk involved in this should be controllable if we had more effectively learnt from our failures. This perspective reflects on some of the learnings of recent years in relation to the causes of attrition. The term Molecular Obesity is introduced to describe our tendency to build potency into molecules by the inappropriate use of lipophilicity which leads to the premature demise of drug candidates. Introduction The road to the successful discovery of new medicines continues to frustrate all those involved. Patients are still in need of effec- tive drugs for many diseases. Payers are increasingly only prepared to pay for innovative rather than derivative drugs. Legislators are demanding that only the very safest possible drugs are licensed. Researchers are equally frustrated in that, despite all the accumulated knowledge from the new technolo- gies, the challenge of successfully navigating through it to find novel drugs seems to get harder rather than easier. In a recent study of the factors contributing to the estimated $1.8bn that it now takes to get a new drug to market, Paul et al. analysed the value and leverage of different components (risk and costs) from target ID, to Lead ID, to Candidate Selection and finally through clinical trial to a new drug.1 It is clear from their sensitivity analysis that the costs of later stage development and, in particular, that of clinical trials, have the highest cost impact of failure for a particular chemical entity. What is also highlighted is that taking the time to get the properties right in a molecular series at the outset does not add hugely to the overall cost, but it does of course, greatly improve the chance of a compound surviving and thus de-risking the propensity to failure in later stages. With current success in the clinical phases being only about 5% (i.e. 95% attrition), if we could only improve our success rate by just 5% then we would actually double the output of pharma and biotech R&D. An understanding of what drives these levels of attrition is therefore fundamental to achieving any reduction in its consequences. As noted by Philip Ball in a Nature News article he wrote under the title of ‘‘Economists need their own uncertainty prin- ciple’’ it is important to distinguish the genuinely predictable scientific activities (‘‘which need maths’’) from the more chaotic activities (‘‘which need experience and intuition’’).2 There is a striking parallel between his commentary on predictability, and lack of it, in economics and the issues we face in drug discovery. In both cases we need to focus the understood and predictable aspect of our sciences on those parts of the surface that are controllable, while also being prepared for (and perhaps even enjoying) the chaotic nature of the rest. Ball also reminds us of Donald Rumsfeld’s much quoted remarks about ‘known knowns, known unknowns and unknown unknowns’. What Rumsfeld neglected to include is the final part of the matrix which is for ‘unknown knowns’; these are those things that are known but have become unknown, either because we have never learnt them, or forgotten about them, or more dangerously chosen to ignore. The aim of this perspective is to look at some of the aspects of medicinal chemistry that have become increasingly known in recent years but often have a tendency to become unknown again! The fundamental challenge in drug discovery Medicinal Chemistry is at the heart of the drug discovery process and the creativity of medicinal chemists in identifying the best leads and then optimising them on a complex landscape of constraints is often the defining characteristic of successful drug discovery campaigns. This process more often resembles an unpredictable journey on a chaotic surface rather than a quanti- tative and predictive science. In addition, despite increased knowledge there is no doubt that our ability to know how to most effectively apply it to navigate what can be a chaotic journey on the multidimensional landscape idealised in Fig. 1 is not getting easier. When such a bounded chaotic system does have some kind of long term pattern but which is clearly not a simple periodic oscillation or orbit the term Strange Attractor is used.3 A strange attractor has non-integer dimensions and is often defined by its apparent chaotic behaviour. Such characteristics are typically sensitivity to initial conditions and the fact that many different end points can result Computational and Structural Chemistry, GSK Medicines Research Centre, Gunnels Wood Rd., Stevenage, SG1 2NY, UK. E-mail: Mike.M. Hann@gsk.com This journal is ª The Royal Society of Chemistry 2011 Med. Chem. Commun., 2011, 2, 349–355 | 349 Dynamic Article LinksC 75 TPSA < 75 TPSA > 75 TPSA < 75 ClogP < 3 0.39 1.08 0.38 0.5 ClogP > 3 0.41 2.4 0.81 2.59 350 | Med. Chem. Commun., 2011, 2, 349–355 This journal is ª The Royal Society of Chemistry 2011 Pu bl is he d on 0 7 M ar ch 2 01 1. D ow nl oa de d on 2 2/ 12 /2 01 4 18 :4 8: 08 . View Article Online One such publication is that from Hughes et al. at Pfizer and considers in vivo tolerability data on 245 compounds that were looked at in multiple assays – see Table 1.7 Compounds that have a ClogP < 3 and TPSA > 75 gave a 6-fold reduction in prevalence of in vivo toxicity versus compounds with ClogP > 3 and TPSA < 75. This reduction rose to a 24-fold reduction for basic compounds. This is often referred to as the Pfizer 3/75 rule and is a clear global sign post to avoiding likely problem compounds. Another example of an analysis that has led to the emergence of a further set of guidelines is that of the GSK 4/400 rule which relates to compounds with a ClogP less than 4 and a MW less than 400 and how on average they have a more favourable ADMET profile.8 The analysis looked at ca. 30,000 neutral, basic, acidic and zwitterionic molecules that have been profiled in multiple physical chemistry and ADMET assays at GSK – see Fig. 3. Another type of analysis which has been widely publicised is that of Leeson and Springthorpe in their seminal 2007 review.5 Among the many interesting observations in this review, they addressed the issue of receptor promiscuity by analysing the data from 2133 compounds that have been profiled in Cerep Bioprint assays – see Fig. 4. Using a promiscuity scale based on the number of Cerep targets that a compound showed activity at >30% when assayed at 10uM, they showed that compounds with a ClogP < 3 showed decreased promiscuity while compounds with ClogP > 4 had increased risks. The risks are also dependent on the charge state of the molecules with bases and quaternary salts having the most risk and acids the least. They have further codified this in terms of a Lipophilic Ligand Efficiency LLE index, defined as pIC50 LogP. A value of greater than 5 is suggested as leading to significant reduction in the risk of toxicity for any given compound. The reason for this can be readily understood when it is remembered that the lipophilicity scales (such as clogP) are logarithmic and therefore an increase of just one unit in clogP means that there is now ten times more compound present in the highly lipophilic cellular membranes. These membranes are the home to many of the critical signalling systems and inappropriate triggering by local high concentra- tions of not very intrinsically potent compounds, can easily lead to unwanted effects leading to potentially toxicological events. While promiscuity of this non-specific type will be detrimental, there may be situations (e.g. for polypharmacology) where some degree of promiscuity is desirable. A further perspective on attrition related parameters is given by a count of the number of aromatic rings. While clearly related in a non-linear manner to lipophilicity, the analysis of this property by Ritchie and MacDonald gives interesting insights which can be summarised as ‘‘the fewer aromatic rings in an oral drug candidate the better’’ with less than three being suggested as an appropriate target number.9 Continuing this theme of the dangers of too much sp2 or aromatic character in molecules, Humblet and colleagues have shown that the survival rate of compounds through the drug discovery process is enhanced by an increase in the fraction of sp3 hybridised carbon atoms and the number of chiral centres present.10 In addition to giving access to a greater diversity of compounds to explore it seems likely that one of the benefits of chirality in a drug is that it leads to increased complexity (and hence potential potency through appropriate complementarity) to a specific target without increasing the molecular weight of ligands.12 Clear evidence that a chiral motif (i.e. increased complexity) is contributing greater molecular recognition can be found by Eudismic analysis within a series.13 Lipophilicity is well known to be the antithesis of solubility and lack of solubility has been a consistent problem for medicinal chemists.19,20 As noted earlier improved formulation methodolo- gies can somewhat mitigate this situation. However relying on formulation to get insoluble compounds on board is likely only to aggravate the body to work harder to eliminate them. The usual response of the body to lipophilic xenobiotics is to try to make them more polar via metabolism so they can be excreted. Medi- cinal chemists are then faced with the need to make their lipophilic and insoluble compounds more metabolically stable to prolong their duration of actions. This can put enormous demands on a compounds profile, especially if once a day dosing is being sought after as the target product profile. Blocked metabolism means the body will need to find more extreme ways of removing the compound, often inducing more high potential and thus intrinsically more reactive and toxic species. It is clear from the above discussion that increasing MW and lipophilicity in our hunt for potency, results in molecules that could be described as obese in that they have become too large and too lipophilic for their own good. This addiction to what I term molecular obesity represents a high risk to the future ‘‘health’’ of the molecule as a drug candidate.21 As with medical Fig. 3 GSK 4/400 rule for neutral molecules for ADMET issues (reproduced with permission from ref. 8). Fig. 4 Promiscuity as function of ClogP (drawn using data supplied by Paul Leeson5). This journal is ª The Royal Society of Chemistry 2011 Med. Chem. Commun., 2011, 2, 349–355 | 351 Pu bl is he d on 0 7 M ar ch 2 01 1. D ow nl oa de d on 2 2/ 12 /2 01 4 18 :4 8: 08 . View Article Online obesity, which is measured by Body Mass Index (BMI), medi- cinal chemists have developed their own indices such as Ligand Efficiency LE (¼ binding affinity/number of heavy atoms)19 and Lipophilic Ligand Efficiency LLE5 to help identify and control the effects of molecular obesity, which have clearly caused the premature death of far too many drug candidates in recent years. Additional indices continue to emerge in the literature, all with the aim of restricting the tendency to Molecular Obesity. One such index is the LELP index, which combines aspects of LE and LLE into a single index by dividing logP by LE. It thus represents the price of ligand efficiency paid for with logP.16 Two excellent recent reviews include further in depth discus- sion of physicochemical related attrition issues and are recom- mended for further reading.26,27 Driving potency through molecular obesity So if excess molecular weight and lipophilicity are not conducive to the ultimate success of compounds in the drug discovery process why is it that we so readily find ourselves at the limits or beyond of what is the statistically safer zone. One of the key drivers that leads to this situation is our perceived need for potency, bordering on an addiction, and the fact that potency can often be most easily gained by increasing the number of interactions our ligands make and/or by increasing lipophilicity. We like potency in our molecules for a number of reasons and it is worth examining why this is. One of the basic tenets of medicinal chemistry is that increasing ligand potency leads to increased specificity and hence to an improved therapeutic index.11 One of the corollaries of our study into molecular complexity and the probability of finding hits is that finding appropriate interactions by increasing matched complexity is essential for optimising weak leads to give increased potency.12 However if complexity is introduced unnecessarily it does nothing to build the specificity rather it can introduce the opportunity for recognition at other targets. High potency is also often perceived to allow reduction in the size of dosage and thus helps reduce the cost of goods. While potency can compensate for low bioavailability in that the small portion of, for example, a poorly absorbed drug that does get into the circulation will at least have a chance of being efficacious if it has high molar potency. However this brings a high risk in that the part of the high dosage that is not being effectively utilised is available to cause off target issues. So while there are many reasons why potency is a good thing, the problem is how we go about achieving it. Again it is the power of looking at larger and more cross-series data than are usually published in a specific medicinal chemistry case history that is revealing. Ladbury and colleagues have shown that the Free Energy of interaction for synthetic ligands correlates well with a ligand’s hydrated apolar surface area (ie lipophilicity) that is buried in the interaction leading to the crystallographically observed complex.14 This phenomenon is even more relevant when the total buried surface area is deconvoluted into the apolar and polar parts, where it is the apolar contributions that domi- nate. This shows that, at least for this data set which is represen- tative of a wide range of complexes typical of drug discovery programmes, we tend to use the easy gains of potency by adding lipophilicity. Thermodynamic considerations in lead optimisation are also prominent in the work of Ferenczy and Keseru. They have introduced the term Size Independent Enthalpic Efficiency (SIHE) to help monitor the contribution of the enthalpy to potency as lead optimisation progresses. Thus monitoring thermodynamic profiles during optimisation should enable a more appropriate balance between enthalpic and entropic potency contributions to be achieved rather than just relying on logP as a surrogate.15 These and other authors have also high- lighted that inappropriate medicinal chemistry can just as easily lead to poor compounds whether the starting point comes from HTS or a fragments approach.16 The problem with the proli- feration of so many ‘‘rules’’ is the trend to slavishly apply them without really understanding their required context for use and subsequent limitations. In other surveys of specific and cross-series data it is also apparent that increasing molecular weight and complexity tend to correlate with increased potency.17 This is consistent with adding either specific or non-specific interactions but is also likely to be a consequence of the predilection of medicinal chemists, who invariably trained as synthetic organic chemists, to build molecules rather than take them apart. Interestingly the equi- valent of retro-synthetic analysis, which is so critical to planning good syntheses, has only recently become more embedded into the medicinal chemistry sphere in terms of fragmenting hits to find the most ligand efficient and smallest critical part.18 A recent study by Waring and colleagues of 9598 AstraZeneca compounds has shown that as molecules are made larger then they need more lipophilicity to be permeable through cell membranes. Thus the apparent twin drivers of potency (mw ¼ more interactions and logP ¼ increased permeability) are seen to be not truly independent variables in relation to bioavailabilty but increasingly linked as compounds get larger.19 In Lipinski’s rule of five, the cut off of MW of 500 is consistent with the experimentally observed upper limit of permeability of compounds through membranes without invoking active trans- port. What Waring’s work now clearly shows is that the space below 500 is not binary, in the sense of being permeable with MW less than 500, but that it has an increasing logP demand as 500 is approached. Another reason why it is all too easy to increase logP in the early stage of drug discovery projects is that if the initial assay is a very specific target assay (eg enzyme or artificially constructed complex) then hits found there are often rapidly progressed down a screening cascade that has a cellular and likely more phenotypic assay facing the nascent leads. In order to gain cellular potency it is all too easy to add lipophilicity to get a compound into the right compartment. This tool compound may have short term benefits of demonstrating cellular efficacy but it is equally too easy to then just forge ahead with this ‘‘fattened ligand’’ in the desire to make further speedy progress towards the project’s next milestones. We should pause at this stage to re-evaluate the properties of the now current lead compounds. Of course, in previous eras of drug discovery when in vivo testing was often much earlier in the discovery process this issue did not exist, because compounds that either showed no activity through lack of bioavailability or toxicity through inappropriate mode of action or side effects were detected and dropped very early. 352 | Med. Chem. Commun., 2011, 2, 349–355 This journal is ª The Royal Society of Chemistry 2011 Pu bl is he d on 0 7 M ar ch 2 01 1. D ow nl oa de d on 2 2/ 12 /2 01 4 18 :4 8: 08 . View Article Online Behavioural addictions in drug discovery While a typical medicinal chemistry publication will set out to show that a very logical process was followed towards achieving the project’s objectives, it is clear that by any objective or subjective measure, the path that a drug discovery project actu- ally follows through a multi-dimensional property space is non- linear. Fig. 1 illustrated this by showing a pathway that could be taken from a start point X1 which has been found by some potency based screening process. From there on, there are numerous pathways (one of which is illustrated) that could be taken to finally reach Xn which is the candidate drug. Whether there is overlap of the zones in any given project as implied in Fig. 1 is only found out by trial and error, although experience with a related target may well inform on such target tractability. We have seen that potency can have an unhealthy connection to molecular obesity, so if we wish to avoid the impact of molecular obesity then we need to ensure that the initial condi- tions in terms of MW and logP are fully explored to find those that are best suited to living with potency as a strange attractor. This is very much the raison d’etre of Fragment Based Drug Discovery – this and other strengths of this approach are high- lighted in Table 2. Another important way to diminish the negative effects of potency is to be more realistic about what level of potency we should aspire to. Again surveys of literature data can help reset expectations. Overington et al.’s analysis of a data set of known drugs shows that the median affinity for current small-molecule drugs is ca. 20 nM (pIC50 ¼ 7.7) – see Fig. 5.24 In an excellent analysis of lead/drug pairs, Perola highlights many of the challenges of successful lead optimisation discussed in this current perspective. The successful drug examples discussed show a mean pIC50 of ca. 8 for the final drugs if the micromolar compound Topetecan is excluded.25 When Topotecan (pKi 5.5) is included in Perola’s full set the mean value is 6.7. Unpublished in house data suggests that for oral drugs the affinity median also shifts to less potency being required (pIC50¼ 6.7). These average levels of potency for successful drugs are considerably lower than the often aspired to pKi or pIC50 values of greater than or equal to 9. Different target classes (eg ion channels vs. GPCRs) will effect the aspirational potency at the outset of a project but as efficacy is, at the end of the day, what is needed, greater emphasis on the factors that can enable overall drug efficacy need to be more to the fore in the earliest stages rather than just potency at the target. Thus, while the desire for potency is understandable, the tendency to choose the most potent compounds in lead selection and then let it remain the primary driver in early stage lead optimisation remains a strong attractor and must be resis- ted.25 The fact that potency is often easy to measure (once the assay is established) can often mean that this is the data most likely first returned to the team. The team (or individual) then tends to react to it by making synthesis decisions without waiting for a fuller profile of data on which to make a more informed decision as to what to make next. So reducing the desire for potency in favour of better ADME properties is one way of subjugating the potency strange attractor and its potentially fatal relationship to molecular obesity. Technology to be able to routinely measure the cellular concentration of ligands in cells of interest would also go a long way to helping to do more effective SAR work in cellular assays. More potent molecules should be more selective because the complexity of interactions at the target of interest reduces the probability of their being effective in interactions at off targets.12 From this perspective it therefore seems that increasing MW should be a good thing in that it leads to selectivity. However we have seen that passage through cell membranes required for cellular activity requires ever increasing amounts of lipophilicity as MW increases. It seems possible that it is this concomitant increase in logP that destroys (by increasing the local concen- tration of the drug in membranes) what selectivity that may have been anticipated to be gained from increased complexity. It should also be remembered that lipophilicity is low grade in its discriminatory powers of molecular recognition, compared to the directionality of, for example, hydrogen bonds. Of the data sets and final potencies discussed by Perola and others, we have no way of knowing whether a better drug could have been found with less potency. This is because the expensive process of drug discovery only allows us to test very few exem- plars in the clinic and if those selected had potency as an early pre-requisite, then the number of less potent but possibly better drugs that we have missed will frustratingly remain an unknowable unknown, although the evidence increasingly points to there being many. Table 2 Some strengths of fragments 1. The combinatorial explosion of chemistry space means that fragments can sample more of the available chemistry space at that level of complexity than is possible with more complex molecules.22 2. At lower complexity there is a higher probability of compounds matching the receptor even though they may be harder to detect. More complex molecules are statistically more likely to have more ‘‘clashes’’ and thus not fit/bind.12 3. Medicinal chemists like to build molecules and so starting small with fragments is the natural feedstock for structure-based design. 4. By starting small and selecting the most ligand efficient compounds more lead-like starting points are found which enhance the chances of successful lead optimisation campaigns. 5. By reducing the number of pharmacophoric elements in the initial lead, only the necessary interactions are built in to the compound as it is optimised. This should help ensure good developability properties of the resulting candidates. This is also relevant if polypharmacology is needed.23 Fig. 5 Potencies of marketed small molecule drugs at their target macromolecule (redrawn with data estimated from ref. 24). This journal is ª The Royal Society of Chemistry 2011 Med. Chem. Commun., 2011, 2, 349–355 | 353 Pu bl is he d on 0 7 M ar ch 2 01 1. D ow nl oa de d on 2 2/ 12 /2 01 4 18 :4 8: 08 . View Article Online The evolution of fit molecules Our mission as medicinal chemists is to maximise the use of what is known, and yet to understand the risks of what is not fully known and then develop strategies to integrate them, whilst always recognising that we are ultimately under the apparently chaotic influence of a strange attractor landscape. While medicinal chemists will always aspire to act as intelligent designers of new molecules, it is clearly the processes of mole- cular evolution that allows the fittest compounds to survive and progress to being worthy of being ultimately labelled as a drug. What allows a compound to survive is fitness and this is clearly only partly understood in any given circumstance, so ensuring we do use what we increasingly understand is necessary but not sufficient for ultimate success. In the same way that this perspective began with Philip Ball’s observations on the similarity of aspects of drug discovery to economics, it seems appropriate to close with an observation on parallels with another science. It is interesting to look at the processes of drug discovery from the point of view of evolu- tionary biology terminology. Table 3 contains a suggested concordance of a number of terms from evolutionary biology with those that we use in drug discovery. The parallels and consequences of the similarities in the table are left for the reader to muse on. However one obvious one is that it is the balance between speciation and extinction that determines the outcomes of biological evolution, and similarly it is the balance of success and failure that will continue to determine the fate of the drug discovery industry. Our ability to improve the odds through understanding how to use more effectively what we do know is a key factor in this. Summary Molecular obesity and its inappropriate use to drive potency and get ligands through membranes have been killing too many drug discovery projects. Starting with the smallest possible lead (ie fragments) and striving to maintain their fitness through the use of various indices is now accepted as a key approach in a more holistic approach to contemporary drug discovery. The absolute need for potency should not be as dominant an attractor as we often allow it to become at the expense of other characteristics of a good drug. There will always be some compounds that make it all the way to drugs and which lie outside of the known preferred space for likely success. However unless truly forced to by circumstances that are fully understood, it is not appropriate to set out with the mentality that my project will be the ‘‘exception that proves the rule’’ as a risk mitigation strategy. Note added in proof A comprehensive review has recently been published on the issues involved in the balance of in vitro potency, ADMET and physicochemical parameter. In particular the authors highlight the poor correlation between in vitro potency and human dose required for efficacy.28 Acknowledgements I would like to thank many present and former colleagues at GSK and colleagues in the wider community involved in drug discovery for their contributions to the ideas expressed in this perspective – while they may not all agree with my views expressed here many have engaged in active debate at meetings. In particular I would like to thank Andrew Leach, Darren Green, Ian Churcher, Andrew Brewster, Paul Gleeson, Andrew Hopkins and Paul Leeson. I am also very grateful for the input of George Keseru at Gideon Richter in Hungary for suggestions on an earlier version of this manuscript and the referees for their helpful suggestions. References 1 S. M. Paul, D. S. Mytelka, C. T. Dunwiddie, C. 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BioSyst., 2006, 2, 429; (b) L. C. Blum and J.-L. Reymond, J. Am. Chem. Soc., 2009, 131, 8732. 23 R. Morphy and Z. Rankovic, Drug Discovery Today, 2007, 12, 156. 24 J. P. Overington, B. Al-Lazikani and A. L. Hopkins, Nat. Rev. Drug Discovery, 2006, 5, 993–996. 25 E. Perola, J. Med. Chem., 2010, 53, 2986. 26 M. J. Waring, Expert Opin. Drug Discovery, 2010, 5(3), 235–248. 27 P. D. Leeson and J. R. Empfield, Annu. Rep. Med. Chem., 2010, 45, 393–407. 28 M. P. Gleeson, A. Hersey, D. Montanari and J. Overington, Nat. Rev. Drug Discovery, 2011, 10, 197–208. This journal is ª The Royal Society of Chemistry 2011 Med. Chem. Commun., 2011, 2, 349–355 | 355 Pu bl is he d on 0 7 M ar ch 2 01 1. D ow nl oa de d on 2 2/ 12 /2 01 4 18 :4 8: 08 . View Article Online Copy with citationCopy as parenthetical citation