PI: Coleman, Kenneth Title: Antibiotics for Recalcitrant Infection

Received: 12/05/2013 FOA: PA10-123 Council: 05/2014

Competition ID: ADOBE-FORMS-B1 FOA Title: NIAID ADVANCED TECHNOLOGY SBIR (NIAID-AT-SBIR [R43/R44])

1 R44 AI112187-01A1 Dual: Accession Number: 3650924

IPF: 10010171 Organization: ARIETIS

Former Number: Department:

IRG/SRG: ZRG1 IDM-U (10)B AIDS: N Expedited: N

Subtotal Direct Costs Animals: Y New Investigator: N

(excludes consortium F&A) Humans: N Early Stage Investigator: N

Year 1: Clinical Trial: N

Year 2: Current HS Code: 10

Year 3: HESC: N

Year 4:

Senior/Key Personnel: Organization: Role Category:

Kenneth Coleman Arietis Corporation PD/PI 

Kim Lewis Northeastern University Consultant

Steven Leonard Northeastern University Consultant

Appendices

Sbc 00009717

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State Application Identifier

Applicant Identifier

1. * TYPE OF SUBMISSION 4. a. Federal Identifier

5. APPLICANT INFORMATION * Organizational DUNS:

* Legal Name:

Department: Division:

* Street1:

Street2:

* City:

* State:

* Country: * ZIP / Postal Code:

Person to be contacted on matters involving this application

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If Revision, mark appropriate box(es).

9. * NAME OF FEDERAL AGENCY:

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10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER:

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2. DATE SUBMITTED

3. DATE RECEIVED BY STATE APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R) 

County / Parish:

Province:

Prefix:

What other Agencies?

Pre-application Application Changed/Corrected Application

Arietis Corporation

650 Albany St.

Boston Suffolk

02118-2518

Dr. Michael

LaFleur

Antibiotics for Recalcitrant Infection

Yes No

USA: UNITED STATES

MA: Massachusetts

R: Small Business

National Institutes of Health

New Resubmission

Renewal Continuation Revision

8. * TYPE OF APPLICATION:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011 

b. Agency Routing Identifier

12. PROPOSED PROJECT:

* Start Date * Ending Date

07/01/2014 06/30/2018

* 13. CONGRESSIONAL DISTRICT OF APPLICANT

MA-008

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

* First Name: Middle Name:

* Last Name: Suffix:

Position/Title:

* Organization Name:

Department: Division:

* Street1:

Street2:

* City:

* Country: * ZIP / Postal Code:

* Phone Number: Fax Number:

* Email:

* State:

County / Parish:

Province:

Prefix:

Coleman

Dr. Kenneth

02118-2518

Boston Suffolk

650 Albany ST

Arietis Corporation

MA: Massachusetts

USA: UNITED STATES

	


 		

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SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

15. ESTIMATED PROJECT FUNDING 16. * IS APPLICATION SUBJECT TO REVIEW BY STATE EXECUTIVE 

ORDER 12372 PROCESS?

a. YES a. Total Federal Funds Requested

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein are 

true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with any resulting 

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administrative penalities. (U.S. Code, Title 18, Section 1001)

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* Email:

* Signature of Authorized Representative * Date Signed

20. Pre-application

* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.

County / Parish:

c. Total Federal & Non-Federal Funds

18. SFLLL or other Explanatory Documentation

Province:

b. Total Non-Federal Funds

Prefix:

0.00

0.00

* I agree

Dr. Michael

LaFleur

Chief Operating Officer

Arietis Corporation

650 Albany St.

Boston Suffolk

MA: Massachusetts

USA: UNITED STATES 02118-2518

Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

DATE:

THIS PREAPPLICATION/APPLICATION WAS MADE 

AVAILABLE TO THE STATE EXECUTIVE ORDER 12372 

PROCESS FOR REVIEW ON:

PROGRAM HAS NOT BEEN SELECTED BY STATE FOR 

REVIEW

PROGRAM IS NOT COVERED BY E.O. 12372; OR

Michael LaFleur 12/05/2013

b. NO

d. Estimated Program Income

	


 		

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424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Face Page------------------------------------------------------------------------------------------ 1

Table of Contents--------------------------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

 Project Summary/Abstract (Description)---------------------------------------- 6

 Public Health Relevance Statement (Narrative attachment)---------------------------------------- 7

 Facilities & Other Resources---------------------------------------- 8

 Other Attachments---------------------------------------- 10

 Sbc 000097171---------------------------------------- 10

Research & Related Senior/Key Person-------------------------------------------------------------------------- 11

 Biographical Sketches for each listed Senior/Key Person---------------------------------------- 13

Research & Related Budget - Year 1---------------------------------------------------------------------------- 22

Research & Related Budget - Year 2---------------------------------------------------------------------------- 25

Research & Related Budget - Year 3---------------------------------------------------------------------------- 28

Research & Related Budget - Year 4---------------------------------------------------------------------------- 31

 Budget Justification---------------------------------------- 34

Research & Related Budget - Cumulative Budget----------------------------------------------------------------- 44

SBIR/STTR Information----------------------------------------------------------------------------------------- 45

 Commercialization Plan---------------------------------------- 47

 Prior SBIR Phase II Awards---------------------------------------- 57

PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------ 58

PHS 398 Specific Research Plan-------------------------------------------------------------------------------- 60

 Introduction---------------------------------------- 61

 Specific Aims---------------------------------------- 62

 Research Strategy---------------------------------------- 63

 Vertebrate Animals---------------------------------------- 75

 Bibliography & References Cited---------------------------------------- 82

 Letters of Support---------------------------------------- 84

 Resource Sharing Plan---------------------------------------- 87

PHS 398 Checklist--------------------------------------------------------------------------------------------- 88

Appendix

Number of Attachments in Appendix: 1

Table of Contents Page 3

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
OMB Number: 4040-0010 

Expiration Date: 08/31/2011

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Performance Sites Page 4

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth

Project/Performance Site Location(s)

Organization Name:

Project/Performance Site Primary Location

Arietis Corporation

650 Albany St

Boston

MA: Massachusetts

USA: UNITED STATES

02118-2518

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

DUNS Number:

* Street1:

* State:

* City: 

Street2:

County:

Province:

* ZIP / Postal Code:

* Country:

* Project/ Performance Site Congressional District: MA-008

Project/Performance Site Location a

Organization Name:

DUNS Number:

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

* ZIP / Postal Code:

* Country:

Province:

* State:

* City: 

Street2:

* Street1:

County:

* Project/ Performance Site Congressional District: 
RESEARCH & RELATED Other Project Information

Yes No

Yes No

Yes No

Yes No

05/29/2013

- 

Yes No

Yes No

Yes No

Yes No

Yes No

1237-Abstract.pdf Add Attachment Delete Attachment View Attachment

1238-Narrative.pdf Add Attachment Delete Attachment View Attachment

1239-Bibliography.pdf Add Attachment Delete Attachment View Attachment

1240-Facilities_Biohazards.pdf Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

Add Attachments Delete Attachments View Attachments

Yes No

OMB Number: 4040-0001 

Expiration Date: 6/30/2016

1. Are Human Subjects Involved?

1.a. If YES to Human Subjects

If yes, check appropriate exemption number.

Is the Project Exempt from Federal regulations?

1 2 3 4 5 6

IRB Approval Date:

Human Subject Assurance Number:

2. Are Vertebrate Animals Used?

If no, is the IRB review Pending?

2.a. If YES to Vertebrate Animals

IACUC Approval Date:

Animal Welfare Assurance Number:

Is the IACUC review Pending?

3. Is proprietary/privileged information included in the application? 

4.a. Does this Project Have an Actual or Potential Impact - positive or negative - on the environment? 

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or 

 environmental impact statement (EIS) been performed?

4.d. If yes, please explain:

5. Is the research performance site designated, or eligible to be designated, as a historic place?

5.a. If yes, please explain:

	


 		

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Other Information Page 5

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth

6. Does this project involve activities outside of the United States or partnerships with international collaborators?

6.a. If yes, identify countries:

6.b. Optional Explanation:

7. Project Summary/Abstract

8. Project Narrative

9. Bibliography & References Cited

10. Facilities & Other Resources

11. Equipment

12. Other Attachments
The goal of the project is to develop a therapeutic capable of sterilizing recalcitrant chronic infections such as 

deep-seated abscess, osteomyelitis, endocarditis, and biofilms of indwelling devices. Many of these infections 

are essentially untreatable and lead to substantial morbidity and mortality. In many cases, recalcitrance of an 

infection is not caused by drug resistance. Rather, a slow-growing biofilm population harbors stationary phase 

and dormant persister cells that are highly tolerant to killing by antimicrobials. When antibiotic concentration 

drops, these cells can grow and repopulate the biofilm. In Gram-positive S. aureus, it appears that a stationary 

culture is made mostly of drug tolerant cells which are virtually insensitive to killing by traditional antibiotics. 

There are many independent, redundant mechanisms of persister formation, and these specialized survivor 

cells do not have a realistic target which could be exploited for drug development. In order to act, all existing 

bactericidal antibiotics require active targets which they corrupt. We reasoned that persisters could be killed if a 

small molecule could simultaneously activate and corrupt a cellular target. We find that acyldepsipeptide 

(ADEP) activates the ClpP protease in dormant persisters, forcing the cell to self-digest. In order to diminish 

resistance development, ADEP was combined with rifampicin. The combination completely sterilized a deepseated biofilm infection of S. aureus in a neutropenic mouse model after a single dose. The best conventional 

antibiotics, alone or in combination, had very little effect. This model emulates the most difficult to treat chronic 

infection in immunocompromised patients. In this project, we will identify the most promising sterilizing 

combinations of drugs. Combinations will be evaluated for PK, PD, and efficacy using a deep-seated 

neutropenic thigh model of MRSA infection, and in an in vivo biofilm tissue-cage model. Once validated, the 

combination therapeutic will enter into preclinical investigation in Phase II, leading to an IND, and subsequent 

clinical trials of the drug. 

Project Description Page 6

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
The goal of the project is to develop a therapeutic capable of curing currently untreatable chronic infections. 

Bacterial pathogens are not susceptible to killing by traditional antibiotics when they are in a non-growing, 

inactive state. This makes it very difficult to treat diseases such as infective osteomyelitis or endocarditis. We 

have identified a compound capable of killing inactive cells, and we will develop this compound into a 

therapeutic. 

Public Health Relevance Statement Page 7

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Laboratory: The Arietis laboratory is located in the Biosquare Discovery and Innovation Center of BU Medical 

School. The 1400 square foot laboratory space and adjacent office space is sufficient for undertaking the work 

on this project. The laboratory is equipped with biosafety cabinets, anaerobic and microaerophilic chambers, 

chemical hoods, shakers, incubators, refrigerators, freezers, microscopes, fluorescent and visible 

spectrophotometers, PCR machines, MicroFill liquid handling dispenser, electroporation device, tissue 

homogenizer, sonicator, spiral plater with colony enumeration system, photo documentation system with transillumination, and small equipment items – gel boxes, balances, water baths, and centrifuges. Common facilities 

adjacent to the suite include cold rooms, centrifuges, autoclaves, glass washers, incubator space and freezers. 

Arietis has also negotiated access to the full suite of core facilities available onsite at the BU Medical Center. 

Core facilities consist of Analytical Instrumentation Core, Biomedical Imaging Center, Biospecimen Archive 

Research Core, BU Clinical and Translational Science Institute, Cellular Imaging Core, Confocal Facility, 

Experimental Pathology Laboratory Service Core, Flow Cytometry Core Facility, High Throughput Screening 

Core, Illumina Sequencing Core Facility, Immunohistochemistry (IHC) Core Facility, LinGA - Linux Genetic 

Analysis Core, Microarray Resource Core Facility, Molecular Genetics Core Facility, Proteomics Core Facility, 

Transgenic Center. More information regarding the specific instrumentation at each core facility can be found 

at http://www.bu.edu/cores/cores/. 

Animal: The animal studies will be performed by Arietis and take place Laboratory Animal Science Center 

(LASC) at Boston University, an AAALAC accredited animal care program located on the medical campus 

comprising Boston University School of Medicine, Boston University School of Public Health, Boston University 

Goldman School of Dentistry and Boston Medical Center. The animal facilities contain approximately 45,780 

sq. feet of animal housing and support space. The Laboratory Animal Science Center is a team of 

administrative, managerial, technical and professional staff committed to the advancement of science in 

collaboration with the research community by promoting the humane care and use of animals used in 

biomedical research and teaching. 

The Laboratory Animal Science Center (LASC) oversees veterinary medical care, animal care services, 

and actively participates in all facility design. Day-to-day care of animals and technical assistance are provided 

by approximately thirty animal caretakers. The care staff is overseen by Operations Managers and Animal 

Care Supervisors. In addition, three veterinary technicians are supervised by a Veterinary Services Manager, 

under the direction of the Attending Veterinarian. Administrative, business and purchasing functions are 

supported by a Business Manager and three support staff. Standard Operating Procedures and reference 

materials are available from the IACUC Office for animal use. The animal health program for all Boston 

University owned laboratory animals is directed by the attending veterinarian, 

, and provided by two full-time veterinarians. All studies involving animals will be performed 

in the Laboratory Animal Sciences Center (LASC). Animals in each LASC room are observed daily for signs of 

illness by the animal technician responsible for providing husbandry. Medical records and documentation of 

experimental use are maintained individually for non-rodents and individually or by cage group for rodents. 

Veterinary technicians under the direction of the attending veterinarian provide routine veterinary medical care 

to all animals. Animal care and use is additionally monitored for training and compliance issues by the Training 

and Compliance Manager. The Boston University Medical Campus Animal Welfare Assurance number is 

.

Computer: The group has PC computers with internet access and support is available. In addition, 

researchers at Arietis have full desktop access to: the Collaborative Drug Discovery database allowing easy 

sharing of chemical structure/properties data, with biological screening data populated by collaborator labs; 

Pipeline Pilot which provides the capability for merging and manipulating data from disparate sources; 

ChemAxon JChem scientific software suite allowing library enumeration, searching, and filtering; 

CambridgeSoft ChemBioOffice; and Statistica analytical software.

Biohazards: S. aureus is a Biosafety Level 2 (BSL-2) microorganism associated with a human disease, for 

which preventive or therapeutic interventions are available. The Arietis Laboratory is BLS-2 certified and all 

personnel receive BSL-2 training from Boston University and are authorized to work with this pathogen. 

Training courses include general laboratory, chemical safety and pathogen specific BSL-2 protocols, which are 

renewed each year. All personnel are monitored annually by the Boston University Occupational Health 

Center, to which any incidents of exposure are reported. To minimize exposure, guidelines detailed in the 

Boston University Safety Manual are followed including that all work is carried out in biological safety cabinets 

(certified annually), all personnel wear personal protective equipment including a laboratory coat, gloves and 

eye protection, and sterilization procedures are followed for the handling of waste: waste cultures are mixed 

Facilities Page 8

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
with 10% fresh bleach for 30 minutes; plastic wear is bagged and sealed within the biological safety cabinet 

before being autoclaved with other biological hazardous waste; glass wear is soaked in 10% fresh bleach for 

30 minutes before further washing; all work surfaces are wiped down with 70% ethanol after working and at the 

end of each day. To clean and contain spills, all personnel are notified to stay away from the area and the spill 

is covered with paper towels, soaked with 10% fresh bleach for 30 minutes before being wiped up from the 

edges towards the center. Safety is monitored by all laboratory members including the PI and Laboratory 

Safety Officer. Review of protocols and procedures is carried out by the Arietis Institutional Biosafety 

Committee to ensure that practices are in compliance with Boston University standards and good laboratory 

practices. All strains are logged in the Arietis Laboratory Database, a copy of which is also held by the Boston 

University Health and Safety department. On-site environmental health and safety consulting services are 

available through Boston University. 

Facilities Page 9

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
SBIR.gov SBC Registration Control ID Form

 SBC CONTROL ID SBC_000097171

 ARIETIS

 BOSTON MA Zip

 FIRM INFORMATION

 Company

 Address 650 Albany St

 City State

 TIN/EIN DUNS 

 Company URL 

 Number of Employees: 

 Is this SBC majority-owned by multiple venture capital operating companies, 

 02118-

No

 hedge funds, or private equity firms?

 What percentage (%) of the SBC is majority-owned by multiple venture capital 0%

 operating companies, hedge funds, or private equity firms?

 SBC CONTROL ID SBC_000097171

Generated June 18, 2013 | U.S. Small Business Association | SBIR.gov

Sbc 000097171 Page 10

Powered by TCPDF (www.tcpdf.org)

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
RESEARCH & RELATED Senior/Key Person Profile (Expanded)

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

PROFILE - Project Director/Principal Investigator

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Position/Title: Department:

Organization Name: Division:

* Street1:

Street2:

* City: County/ Parish:

* State: Province:

* Country: * Zip / Postal Code:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Project Role: Other Project Role Category:

*Attach Biographical Sketch

Attach Current & Pending Support

Dr. Kenneth

Coleman

Arietis Corporation

650 Albany ST

Boston Suffolk

MA: Massachusetts

USA: UNITED STATES 02118-2518

PD/PI

1234-Biosketch_KC.pdf Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment

Degree Type:

Degree Year:

View Attachment

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Position/Title: Department:

Organization Name: Division:

* Street1:

Street2:

* City:

Professor

Northeastern University

134 Mugar Hall

Biology

USA: UNITED STATES

Kim

Lewis

360 Huntington Ave

Boston

MA: Massachusetts

02115-0000

County/ Parish:

* State:

* Country: * Zip / Postal Code:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Project Role: Consultant Other Project Role Category:

1

Degree Type:

Degree Year:

*Attach Biographical Sketch Add Attachment Delete Attachment View Attachment

Attach Current & Pending Support Add Attachment Delete Attachment View Attachment

1235-Biosketch Lewis.pdf

PROFILE - Project Director/Principal Investigator

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name:

Position/Title: Department:

Division:

* Street1:

Street2:

* City:

Dr. Kenneth

Coleman

Arietis Corporation

650 Albany ST

Boston Suffolk

MA: Massachusetts

USA: UNITED STATES 02118-2518

PD/PI

1234-Biosketch_KC.pdf Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment

County/ Parish:

Province:

* Country:

* State:

* Zip / Postal Code:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Project Role: Other Project Role Category:

Degree Type:

Degree Year:

Attach Current & Pending Support

*Attach Biographical Sketch

View Attachment

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name:

Position/Title: Department:

Division:

* Street1:

Street2:

* City:

Professor

Northeastern University

134 Mugar Hall

Biology

USA: UNITED STATES

Kim

Lewis

360 Huntington Ave

Boston

MA: Massachusetts

02115-0000

County/ Parish:

* Country:

* State:

1

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Key Personnel Page 11

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth

* Zip / Postal Code:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Project Role: Consultant Other Project Role Category:

Degree Type:

Degree Year:

*Attach Biographical Sketch

Attach Current & Pending Support
RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Senior/Key Person

Middle Name:

* Last Name: Suffix:

Prefix: * First Name:

Organization Name:

Position/Title: Department:

Division:

* Street1:

Street2:

* City: County/ Parish:

Province:

* Country:

* State:

* Zip / Postal Code:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

Assistant Professor

Northeastern University

Pharmacy

USA: UNITED STATES

Steven

Leonard

140 The Fenway, R225

Boston

MA: Massachusetts

02115-0000

* Project Role: Consultant Other Project Role Category:

2

Degree Type:

Degree Year:

*Attach Biographical Sketch 1236-Leonard_Biosketch.pdf Add Attachment Delete Attachment View Attachment

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Key Personnel Page 12

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME 

Coleman, Kenneth 

eRA COMMONS USER NAME (credential, e.g., agency login) 

POSITION TITLE 

Principal Investigator

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and 

residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable) MM/YY FIELD OF STUDY

University of London, London, UK 

Univ. of Nottingham, Nottingham, UK

B.Sc. 

Ph.D.

1970

1974

Microbiology/Chemistry

Microbiology

A. Personal Statement

I have a broad background in microbiology and chemistry, with specific training and expertise in key research 

areas for this application, and have worked in the anti-infective area in small and large pharmaceutical 

companies for the past 35 years. For most of that time I have been focused on lead optimization of antibacterial 

and antifungal compounds, but I have also been a member of development project teams and have managed 

numerous discovery and development projects myself. While working in the UK, I held a Home Office 

experimental animal license for 20 years and ran many in vivo anti-infective models, including the murine 

staphylococcal thigh model and the tissue cage model. Due to a strong anti-vivisection group in the UK, 

company policy discourages publication of in vivo work, so there is little evidence of this capability in my 

publication list. While at GSK, I contributed to the development of the two clavulanic acid drugs, Augmentin 

and Timentin, and to the quinolone, gemifloxacin. 

In my last position, as Chief Scientific Officer of Novexel SA (Romainville, France) I oversaw the biology and 

chemistry research departments and the Safety Assessment department of the company and was on the steering 

team for three development projects, two antibacterial (NXL103 and NXL104) and one antifungal (NXL201). 

We advanced NXL104 (avibactam), a novel β-lactamase inhibitor, through a successful Phase II clinical trial, 

after which the Company was acquired by Astra Zeneca. 

I joined Arietis in 2010. We are an early startup Biotech focused on narrow-spectrum agents and compounds 

capable of treating dormant pathogens in chronic infections. An ADEP4 combination is our first candidate for a 

systemic sterilizing antimicrobial. It comes out of a successful collaboration with Kim Lewis’s group at NU, and

I feel that we have a good chance to develop this into a therapeutic for curing currently untreatable infections. 

B. Positions and Honors

Positions and Employment

1971 - 1974 Lecturer in Microbiology, Trent Polytechnic, Nottingham, UK 

1974 - 2001 Assistant Director, SmithKline Beecham Pharmaceuticals 

2001 - 2008 Director, Anti-Infective Biosciences, AstraZeneca R&D Boston 

2008 - 2010 Chief Scientific Officer, Novexel SA, Romainville, France 

2010 - Chief Scientific Officer, Arietis Corp., Boston, MA 

2004 – Editorial Board, Antimicrobial Agents & Chemotherapy 

Biosketches Page 13

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
2009 – 11 CLSI Subcommittee on Susceptibility Testing of Human Mycoplasmas 

C. Selected Peer-reviewed Publications

1. K. COLEMAN, D. R. J. GRIFFIN AND PAULINE A. UPSHON. (1991). Pharmacokinetic studies and renal dehydropeptidase 

stability of the new ß-lactamase inhibitor BRL 42715 in animals. Antimicrobial Agents and Chemotherapy, 35: 1748 - 1752. 

2. K. COLEMAN, M. ATHALYE, M. DAVISON, D. PAYNE, C. PERRY & I. CHOPRA. (1993). Bacterial resistance mechanisms 

as therapeutic targets. Journal of Antimicrobial Chemotherapy, 33: 1091 - 1116. 

3. K. COLEMAN. (1995). An update on ß-lactamases and ß-lactamase inhibitors. Expert Opinion in Investigational Drugs, 4:

693 – 704

4. J. BROSKEY, K. COLEMAN et al. (2000). Efflux and target mutations as quinolone resistance mechanisms in clinical 

isolates of S. pneumoniae. Journal of Antimicrobial Chemotherapy, 45, S1: 95 – 99.

5. K. COLEMAN. (2004). Recent Advances In The Treatment Of Gram-Positive Infections. Drug Discovery Today: Therapeutic

Strategies, . 1: 455 – 460 

6. K. COLEMAN. (2006). Extending The Life Of -Lactam Antibiotics: New -Lactamase Inhibitors. Drug Discovery

Today: Therapeutic Strategies, . 3: 183-188

7. M. BLACK and K. COLEMAN. (2009). New inhibitors of bacterial topoisomerase GyrA/ParC subunits. Current Opinion In

Investigational Drugs, 10 (8), 804-810.

8. K. COLEMAN. (2011). Diazabicyclooctanes (DBOs): a potent new class of non-β-lactam β-lactamase inhibitors. Current

Opinion in Microbiology. 14: 550-555. 

9. LAFLEUR MD, SUN L, LISTER I, KEATING J, NANTEL A, LONG L, GHANNOUM M, NORTH J, LEE RE, COLEMAN K, 

DAHL T, LEWIS K. (2013). Potentiation of azole antifungals by 2-adamantanamine. Antimicrob Agents Chemother. 57:3585-

92. 

 10. CONLON, B. P., E. S. NAKAYASU, L. E. FLECK, M. D. LAFLEUR, V. M. ISABELLA, K. COLEMAN et al. (2013) Activated 

ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503: 365-370.

D. Research Support (ongoing or completed in past 3 years)

06/15/2012-05/31/2014. Compounds to treat Helicobacter pylori infection 

Principal Investigator: Dr Ken Coleman (Dr R. E. Lee Co-Investigator) 

ARIETIS CORP 

Agency: NIH 1 R43AI098327-01A1 

In this study we propose to develop novel and selective anti H. pylori therapeutics.

07/01/2012-06/30/2016. Narrow-spectrum Agents Acting against Helicobacter pylori

Principal Investigator: Dr Ken Coleman 

ARIETIS CORP 

Agency: NIH 1R44AI102452-01

In this study, we will develop our lead series against H. pylori. 

Biosketches Page 14

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Program Director/Principal Investigator (Last, First, Middle): Lewis, Kim 

NAME

Kim Lewis, Ph.D.

POSITION TITLE

University Distinguished Professor

eRA COMMONS USER NAME Director, Antimicrobial Discovery Center

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as 

nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

YEAR(s) FIELD OF STUDY

Moscow University, Moscow, USSR B.S. 1976 Biology

Moscow University, Moscow, USSR Ph.D. 1980 Biochemistry

A. Personal Statement 

The main focus of my research is on antimicrobial tolerance which limits the ability of antibiotics to sterilize 

an infection; and on antimicrobial drug discovery. Pathogen eradication is especially important for chronic 

infections such as those formed by biofilms, where the effectiveness of the immune system is limited. In 

the area of tolerance, my laboratory discovered persister cells in microbial biofilms and showed that this is 

the main culprit of recalcitrance. In recent years, we identified a number of genes involved in persister 

formation, and established detailed molecular mechanisms of dormancy in E. coli governed by the toxin 

components of toxin/antitoxin modules HipBA and TisAB. 

My group has been involved in antimicrobial drug discovery for over a decade, and the program started 

with the discovery of synergistically-acting antimicrobials from medicinal plants. We found that many plant 

antimicrobials are effectively extruded by bacterial multidrug resistance pumps (MDRs). Plants solve the 

problem by simultaneously producing MDR inhibitors. In collaboration with Dr. Slava Epstein, we 

developed a method to grow uncultured bacteria, and identfified the first class of growth factors, 

siderophores, for these organisms. These approaches are being used by scientists from NovoBiotic to 

obtain novel secondary metabolites. My group together with Dr. Fred Ausubel developed an HTS 

approach using live infected animals, the worm C. elegans which is leading to identification of compounds 

that are missed by traditional in vitro screens. 

 The proposed project is a culmination of a decade-long effort by my group to identify a realistic 

approach to eradicate persisters. What we have learned over that decade is that persisters evolved to 

survive conventional antibiotics that hit specific targets. The targets in persisters are inactive, and the 

pathways of dormancy are redundant. It appears that acyldepsipeptides evolved to kill both growing and 

dormant bacteria by activating a protease, forcing cells to self-digest. We will use nature’s elegant solution 

to the dormancy problem as a basis for developing a sterilizing therapeutic. 

B. Positions and Honors: 

From: To: Title: Institution:

7/1/01 Present Professor Northeastern University

4/1/97 6/31/01 Research Assoc. Professor Biotechnology Center, Tufts University

9/94

7/88

1/88

3/31/97

8/94

1/88

Associate Professor

Assistant Professor

Assistant Professor

Medical & Research Technology, UMAB

Department of Biology, MIT

Applied Biology, MIT (Department was disbanded)

7/87 1/88 Research Associate Univ. of Wisconsin, Madison

4/84 7/87

Biographical Sketches for each listed Senior/Key Person 2 Page 15

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth

Lost Academic position after applying to emigrate to the USA

6/79 3/84 Senior Researcher Moscow University

6/76 6/79 Researcher Moscow University

Nobel Conference “Biofilm formation, its clinical impact and potential treatment”, Karolinska Institutet, 

Stockholm, 2013 

Leading Scientists Seminar Series, Imperial College London, 2013 
Program Director/Principal Investigator (Last, First, Middle): Lewis, Kim

Lester O. Krampitz Lecture, Case Western Reserve Univesity, 2013 

Keynote Lecture, Lyme Disease Association annual meeting, Philadelphia, 2012 

Fellow, American Academy of Microbiology, 2011 

University Distinguished Professor, Northeastern University, 2011 

Keynote lecture, Belgian Society for Microbiology, Brussels, 2011 

Speaker and Panelist, The National Academies Forum on Microbial Threat, Washington, DC 2011 

Speaker and Panelist, the NIAID/FDA/IDSA Public Workshop on Antibacterial Resistance and Diagnostic 

Device and Drug Development Research for Bacterial Diseases, Washington, DC 2010 

Speaker and Panelist, The National Academies Forum on Antibiotic Resistance, Washington, DC 2010 

NIH Director’s Transformative Grant Award, 2009 

Keynote Lecture, ASM Educational Meeting (ASMCUE), Fort Collins, CO 2008 

Chair, NIAID-BARDA Application of Platform Technologies for the Development of Therapeutics for Biodefense 

study section, 2008 

Chair, NIH Roadmap study section, High Throughput Screening in the Molecular Libraries Screening Centers 

Network, 2008 

Convener, ICAAC symposium, Antimicrobial Tolerance: Persisters, Biofilms, And Infectious Disease, 

Washington, DC, 2008 

Lecturer, Harvard University Microbial Science Initiative, 2006 

Member, Faculty of 1000, Pharmacology & Drug Discovery section, 2006 - 

Division A (Antimicrobial Chemotherapy) Lecture, ASM General Meeting, Atlanta, Georgia, 2005 

NIH study section member, Drug Discovery and Mechanisms of Antimicrobial Resistance, 2004 –2006

Distinguished Research Fellow, Northeastern University, 2004 

Convener, ASM General Meeting, New Orleans, 2004 

Expert Lecture, V European Congress on Chemotherapy and Infections, Rhodes, Greece, 2003 

Convener, ASM General Meeting, Washington, DC, 2003 

Keynote lecture, Wind River Conference on Prokaryotic Biology, Estes Park, CO, 2001 

Convener, Society for Industrial Microbiology Annual Meeting, Saint Louis, 2001 

Convener, Society for Industrial Microbiology Annual Meeting, San Diego, 2000 

Symposial Lecture at the Society for General Microbiology Meeting, Leeds, 1999 

Chair, MDR Colloquium, 9th European Bioenergetics Meeting (EBEC), Louvain, Belgium, 1996 

Convener, ASM General Meeting, Washington, DC, 1995 

MIT C.E. Reed Faculty Initiative Award for an innovative research project. Competitively awarded to 1 Faculty 

member among the School of Science and the School of Engineering annually, 1992 

INVENTIONS: 

1. Solvent-resistant microorganisms. Klibanov, A. M., Lewis. K., Ferrante, A., Coyle, C. L., Zylstra, G., Logan, 

M. S. P., Grossman, M. J. US Patent #5,807,735. Awarded 9.15.98. 

2. Culturing cells in presence of amphipathic weak bases and/or cations and multiple drug resistance inhibitor 

containing reserpine. Lewis, K., Hsieh, P-C. US Patent #6,410,041. Awarded 6.25.02. 

3. Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon. 

Lewis, K., Epstein, S.S., and Kaeberlein, T. US Patent #7,011,957. Awarded March 14, 2006. Licensed to 

NovoBiotic Pharmaceuticals, LLC.

Biographical Sketches for each listed Senior/Key Person 2 Page 16

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Program Director/Principal Investigator (Last, First, Middle): Lewis, Kim 

4. Antimicrobial polymeric surfaces. Klibanov, A.M., Lewis, K., Tiller, J., Liao, C-J. US 10/123,860 (Patent 

pending). Licensed to Pulmatrix, Inc. 

C. Selected publications (of 90 total). 

Most relevant to the current application. 

Schumacher, M.A., Piro, K.M., Xu, W., Hansen, S., Lewis, K., and Brennan, R.G. 2009. Molecular 

mechanisms of HipA mediated multidrug tolerance and its neutralization by HipB. Science 323:396-401. 

Dörr, T., Vulić, M., and Lewis, K., 2010. Ciprofloxacin causes persister formation by inducing the TisB toxin 

in Escherichia coli. PLoS Biol 8(2): e1000317.

Mulcahy, L.R., Burns, J.L., Lory, S., and Lewis, K. 2010. Emergence of Pseudomonas aeruginosa strains 

producing high levels of persister cells in patients with cystic fibrosis. J. Bacteriol. 192: 6191–6199. 

Keren, I., Wu, Y., Innocencio, J., Mulcahy, L., and Lewis, K. 2013. Killing by bactericidal antibiotics does 

not depend on reactive oxygen species. Science 339: 1213-1216.

Conlon, B.P., Nakayasu, E.S., Fleck, L.E., LaFleur, M.D., Isabella, V.M., Coleman, K., Leonard, S.N., 

Smith, R.D., Adkins, J.N. and Lewis, K. 2013. Protease activation kills persisters and eradicates a chronic 

biofilm infection. Nature 503: 365-370.

Additional recent publications of importance to the field. 

Kaeberlein, T., Lewis*, K., and Epstein*, S.S. (*Equal contribution) 2002. Isolating "uncultivable" 

microorganisms in pure culture using a simulated natural environment. Science 296:1127-1129.

Keren, I., Shah, D., Spoering, A., Kaldalu, N., and Lewis, K. 2004. Specialized persister cells and the 

mechanism of multidrug tolerance in Escherichia coli. J. Bacteriol. 186:8172-8180. 

Hansen, S., Lewis, K., and Vulic, M. 2008. Role of global regulators and nucleotide metabolism in 

antibiotic tolerance in Escherichia coli. Antimicrob. Agents Chemother. 52:2718-2726.

Correia ,F.F., D'Onofrio, A., Rejtar, T., Li, L., Karger, B.L., Makarova, K., Koonin, E.V., and Lewis, K. 2006. 

Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. 

J. Bacteriol. 188:8360-8367. 

Moy, T.I., Ball, A., Anklesaria, Z., Casadei, G., Lewis, K., Ausubel, F.M. 2006. Identification of novel 

antimicrobials using a live-animal infection model. Proc. Natl. Acad. Sci. USA. 103:10414-10419. 

D’Onofrio, A., Crawford, J.M., Stewart, E.J., Witt, K., Gavrish, E., Epstein, S., Clardy, J., and Lewis, K. 

2010. Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chem. & Biol. 17: 

254–264.

Lafleur, M.D., Qi, Q., and Lewis, K. 2010. Patients with long-term oral carriage harbor high-persister 

mutants of C. albicans. Antimicrob. Agents Chemother. 54:39-44. 

Keren, I., Minami, S., Rubin, E., and Lewis, K. 2011. Characterization and transcriptome analysis of 

Mycobacterium tuberculosis persisters. mBio 2: e00100-11. 

Wu, Y., Vulić, M., Keren, I., and Lewis, K. 2012. Role of oxidative stress in persister tolerance. Antimicrob.

Agents Chemother. 56:4922-4026.

LaFleur, M., Sun, L., Lister, I., Keating, J., Nantel, A., Long, L., Ghannoum, M., North, J., Lee, R., Coleman, 

K., Dahl, T., and Lewis, K. (2013) Potentiation of azole antifungals by 2-adamantanamine. Antimicrob. Agents 

Chemother. 57:3585-3592.

Lewis, K. (2013) Platforms for antibiotic discovery. Nat. Rev. Drug Disc. 12: 371–387.

D. Current Research Support/projects completed in the last 3 years: 

Current: 

Transformative (T-RO1) 1R01AI085585-01 (K. Lewis, P.I.) NIH 09/25/2009 – 08/31/2014 

Super-persistent cells and the paradox of untreatable infections 

The aim of the project is to test whether bacteria form super-persistent cells in vivo responsible for 

recalcitrance of disease. 

Biographical Sketches for each listed Senior/Key Person 2 Page 17

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Program Director/Principal Investigator (Last, First, Middle): Lewis, Kim 

1 R01 AI076372-01A2 (K. Lewis, P.I.) NIH 2/2/2009- 2/1/2014

A synergy-based therapy against C. difficile

The aim of the project is to develop a therapeutic against C. difficile based on a combination antimicrobial.

Completed: 

1R01AI085005-01A1 (K. Lewis, P.I.) NIH 07/01/2010 - 06/31/2013 

A High-Throughput screen for specific anti-M. tuberculosis compounds 

The aim of the project is to develop a screen for identifying natural product antimicrobials acting against M. 

tuberculosis. 

1R01HG005824-01 (K. Lewis, P.I.) NIH 07/01/2010 - 06/31/2013

Culturing uncultivatable gut microorganisms

The aim of the project is to develop a high-throughput method to culture gut microorganisms.

3R01 GM061162 -05A1 (K. Lewis, P.I.) NIH 4/1/2008- 3/31/2012

A genomics approach to drug tolerance

The aim of the project is to identify the complement of persister genes in E. coli.

3R01GM061162-10S1 (K. Lewis, P.I.) NIH 9/30/2009 - 8/31/2011

A genomics approach to drug tolerance (ARRA competitive revision)

The aim of the project is to identify the mechanism of action of the TisB persister protein.

K. Lewis, P.I.; E. Rubin, Harvard Medical 

School, Co-PI. Bill & Melinda Gates 

Foundation

11/01/2007 – 10/31/2010

Isolation and properties of M. tuberculosis persisters.

Biographical Sketches for each listed Senior/Key Person 2 Page 18

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME

Leonard, Steven Nathaniel

POSITION TITLE

Assistant Professor of Pharmacy Practice

eRA COMMONS USER NAME (credential, e.g., agency login)

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and 

residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable) MM/YY FIELD OF STUDY

Purdue University B.S. 05/03 Pharmacy

Purdue University Pharm.D. 12/04 Pharmacy

St. Vincent Hospital Residency 06/06 Pharmacy Practice

Wayne State University Fellowship 07/08 Infectious Disease

A. Personal Statement 

The goal of this project is to develop the antibacterial ADEP for the treatment of S. aureus infections. During 

the first phase in vitro testing using an in vitro pharmacokinetic/pharmacodynamic (PK/PD) hollow fiber 

infection model will be employed to find the best companion antibiotic for ADEP. It is during this phase that my 

expertise will be utilized. I have extensive experience using a variety of PK/PD models, including the hollow 

fiber model, to test a variety of antibiotics and combinations of antibiotics. This experience includes preliminary 

work on ADEP with Arietis and theLewis lab. 

B. Positions and Honors 

Positions and Employment

2006-2008 Fellow, Anti-infective Research Laboratory, Wayne State University, Detroit, MI 

2008-Present Assistant Professor, Department of Pharmacy Practice, Northeastern University, Boston, MA 

2008-Present Infectious Diseases Pharmacist, Brigham and Women’s Hospital, Boston, MA

Professional Memberships

2006-Present American Society for Microbiology 

2006-Present Society of Infectious Diseases Pharmacists 

2007-Present American College of Clinical Pharmacy 

2008-Present American Association of Colleges of Pharmacy 

2011-Present European Society of Clinical Microbiology and Infectious Diseases 

Honors

2010 Faculty Inductee, Rho Chi Honor Society, Beta Tau Chapter, Northeastern University 

2011 Northeastern University School of Pharmacy Teacher of the Year 

Biographical Sketches for each listed Senior/Key Person 3 Page 19

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
C. Selected Peer-reviewed Publications (Selected from 23 total) 

1. Rose WE, Leonard SN, Sakoulas G, Kaatz GW, Zervos MM, Sheth AA, Carpenter CF, Rybak MJ. 

Daptomycin Activity against Staphylococcus aureus following Vancomycin Exposure in an In Vitro 

Pharmacodynamic Model with Simulated Endocardial Vegetations. Antimicrob Agents Chemother

2008;52(3):831-6. PMID: 17999971.

2. LaPlante KL, Leonard SN, Andes DR, Craig WA, Rybak MJ. Activities of clindamycin, daptomycin, 

doxycycline, linezolid, trimethoprim-sulfamethoxazole, and vancomycin against community-associated 

methicillin-resistant Staphylococcus aureus with inducible clindamycin resistance in murine thigh infection 

and in vitro pharmacodynamic models. Antimicrob Agents Chemother 2008;52(6):2156-62. PMID: 

18411321.

3. Leonard SN, Cheung CM, Rybak MJ. Activities of ceftobiprole, linezolid, vancomycin, and daptomycin 

against community-associated and hospital-associated methicillin-resistant Staphylococcus aureus. 

Antimicrob Agents Chemother 2008;52(8):2974-6. PMID: 18519721.

4. Rose WE, Leonard SN, Rybak MJ. Evaluation of daptomycin pharmacodynamics and resistance at 

various dosage regimens against Staphylococcus aureus isolates with reduced susceptibilities to 

daptomycin in an in vitro pharmacodynamic model with simulated endocardial vegetations. Antimicrob 

Agents Chemother 2008;52(9):3061-7. PMID: 18591272. 

5. Rybak MJ, Leonard SN, Rossi KL, Cheung CM, Sadar HS, Jones RN. Characterization of vancomycinheteroresistant Staphylococcus aureus from the metropolitan area of Detroit, Michigan, over a 22-year 

period (1986 to 2007). J Clin Microbiol 2008;46(9):2950-4. PMID: 18632899. 

6. Leonard SN, Kaatz GW, Rucker LR, Rybak MJ. Synergy between gemifloxacin and 

trimethoprim/sulfamethoxazole against community-associated methicillin-resistant Staphylococcus aureus. 

J Antimicrob Chemother 2008;62(6):1305-10. PMID: 18801920.

7. Leonard SN, Rybak MJ. Evaluation of vancomycin and daptomycin against methicillin-resistant 

Staphylococcus aureus and heterogeneously vancomycin-intermediate S. aureus in an in vitro 

pharmacokinetic/pharmacodynamic model with simulated endocardial vegetations. J Antimicrob 

Chemother 2009;63(1):155-160. PMID: 18984644. 

8. Rose WE, Leonard SN, Rossi KL, Kaatz GW, Rybak MJ. Impact of inoculum and heterogeneous 

vancomycin intermediate Staphylococcus aureus (hVISA) on vancomycin activity and emergence of VISA 

in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 2009;53(2):805-7. PMID: 

19015334.

9. Leonard SN, Rossi KL, Newton KL, Rybak MJ. Evaluation of the Etest GRD for the detection of 

Staphylococcus aureus with reduced susceptibility to glycopeptides. J Antimicrob Chemother

2009;63(3):489-92. PMID: 19136530.

10. Leonard SN, Vidaillac C, Rybak MJ. Activity of Telavancin Against Staphylococcus aureus of Varying 

Vancomycin Susceptibilities in an In Vitro Pharmacokinetic/Pharmacodynamic Model with Simulated 

Endocardial Vegetations. Antimicrob Agents Chemother 2009;53(7):2928-2933. PMID: 19414568.

11. Vidaillac C, Leonard SN, Rybak MJ. In Vitro Activity of Ceftaroline Against Methicillin-Resistant 

Staphylococcus aureus and heterogeneous Vancomycin-Intermediate S. aureus Using a Hollow Fiber 

Model. Antimicrob Agents Chemother 2009;53(11):4712-7. PMID: 19738009. 

12. Leonard SN, Szeto YG, Zolotarev M, Grigoryan IV. Comparative In Vitro Activity of Telavancin, 

Vancomycin, and Linezolid against Heterogeneously Vancomycin Intermediate Staphylococcus aureus. Int 

J Antimicrob Agents 2011;37(6):558-561. PMID: 21497067.

13. Leonard SN. Synergy between Vancomycin and Nafcillin against Staphylococcus aureus in an In Vitro 

Pharmacokinetic/Pharmacodynamic Model. PLoS One 2012;7(7):e42103. PMID: 22848719.

14. Leonard SN, Rolek KM. Evaluation of the Combination of Daptomycin and Nafcillin against VancomycinIntermediate Staphylococcus aureus. J Antimicrob Chemother 2013;68(3):644-7. PMID: 23152482.

15. Leonard SN, Supple ME, Gandhi RG, Patel MD. Comparative Activity of Telavancin Combined with 

Nafcillin, Imipenem, and Gentamicin against Staphylococcus aureus. Antimicrob Agents Chemother

2013;57(6):2678-83. PMID: 23545527.

Biographical Sketches for each listed Senior/Key Person 3 Page 20

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
D. Research Support 

Completed Research Support

Astellas Pharma and Theravance Inc. 8/2011-8/2012 

Comparative Activity of Telavancin Combined with Nafcillin, Imipenem, and Gentamicin against 

Staphylococcus aureus 

Role: PI 

Cubist Pharmaceuticals 8/2011-6/2012 

Daptomycin combined with nafcillin against vancomycin intermediate Staphylococcus aureus. 

Role: PI 

Astellas Pharma 4/2010-12/2010 

Evaluation of Telavancin, Vancomycin, and Linezolid against Heterogeneously Glycopeptide Intermediate 

Staphylococcus aureus (hGISA). 

Role: PI 

Northeastern University Proposal Development Grant 6/2009-5/2010 

Evaluation of the Combination of Nafcillin and Vancomycin Against Methicillin Resistant Staphylococcus 

aureus with Reduced Susceptibility to Glycopeptides. 

Role: PI 

Biographical Sketches for each listed Senior/Key Person 3 Page 21

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
* Last Name

RESEARCH & RELATED BUDGET - SECTION A & B, BUDGET PERIOD 1

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* Project Role Base Salary ($)

* Fringe 

Benefits ($) * Funds Requested ($)

9.

8.

7.

6.

5.

4.

3.

2.

1.

Total Funds requested for all Senior Key Persons in the attached file

Total Senior/Key Person

Additional Senior Key Persons:

B. Other Personnel

A. Senior/Key Person

* Number of 

Personnel * Project Role

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

Prefix * First Name Middle Name Suffix

* Fringe 

Benefits ($) * Funds Requested ($)

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

RESEARCH & RELATED Budget {A-B} (Funds Requested)

* ORGANIZATIONAL DUNS:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Project Subaward/Consortium

3

Delete Entry

Dr. Kenneth Coleman PD/PI 1.20

1 Senior Scientist 3.00

1 Research Associate 12.00

1 Research Assistant 7.80

Arietis Corporation

07/01/2014 06/30/2015 Budget Period 

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Detailed Budget - Year 1 Page 22

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment item * Funds Requested ($)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11. Total funds requested for all equipment listed in the attached file

D. Travel

1. Domestic Travel Costs ( Incl. Canada, Mexico and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

Total Equipment

E. Participant/Trainee Support Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

Number of Participants/Trainees Total Participant/Trainee Support Costs

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {C-E} (Funds Requested)

RESEARCH & RELATED BUDGET - SECTION C, D, & E, BUDGET PERIOD 1

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

Additional Equipment:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2014 06/30/2015

Arietis Corporation

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Detailed Budget - Year 1 Page 23

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
F. Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

8.

9.

10.

Total Other Direct Costs

G. Direct Costs

Total Direct Costs (A thru F)

H. Indirect Costs Indirect Cost 

Rate (%)

Indirect Cost 

Base ($)

1.

2.

3.

4.

Cognizant Federal Agency

I. Total Direct and Indirect Costs

Total Direct and Indirect Institutional Costs (G + H)

J. Fee

K. * Budget Justification

Indirect Cost Type

Funds Requested ($)

Funds Requested ($)

* Funds Requested ($)

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

Total Indirect Costs

(Only attach one file.)

(Agency Name, POC Name, and POC Phone Number)

1252-Budget.pdf

Hollow fiber

F&A 40.00

RESEARCH & RELATED BUDGET - SECTION F-K, BUDGET PERIOD 1

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2014 06/30/2015

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Detailed Budget - Year 1 Page 24

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
* Last Name

RESEARCH & RELATED BUDGET - SECTION A & B, BUDGET PERIOD 2

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* Project Role Base Salary ($)

* Fringe 

Benefits ($) * Funds Requested ($)

9.

8.

7.

6.

5.

4.

3.

2.

1.

Total Funds requested for all Senior Key Persons in the attached file

Total Senior/Key Person

Additional Senior Key Persons:

B. Other Personnel

A. Senior/Key Person

* Number of 

Personnel * Project Role

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

Prefix * First Name Middle Name Suffix

* Fringe 

Benefits ($) * Funds Requested ($)

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

RESEARCH & RELATED Budget {A-B} (Funds Requested)

* ORGANIZATIONAL DUNS:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Project Subaward/Consortium

3

Delete Entry

Dr. Kenneth Coleman PD/PI

3 Senior Scientists 36.00

Arietis Corporation

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Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment item * Funds Requested ($)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11. Total funds requested for all equipment listed in the attached file

D. Travel

1. Domestic Travel Costs ( Incl. Canada, Mexico and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

Total Equipment

E. Participant/Trainee Support Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

Number of Participants/Trainees Total Participant/Trainee Support Costs

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {C-E} (Funds Requested)

RESEARCH & RELATED BUDGET - SECTION C, D, & E, BUDGET PERIOD 2

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

Additional Equipment:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2015 06/30/2016

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Detailed Budget - Year 2 Page 26

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
F. Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

8.

9.

10.

Total Other Direct Costs

G. Direct Costs

Total Direct Costs (A thru F)

H. Indirect Costs Indirect Cost 

Rate (%)

Indirect Cost 

Base ($)

1.

2.

3.

4.

Cognizant Federal Agency

I. Total Direct and Indirect Costs

Total Direct and Indirect Institutional Costs (G + H)

J. Fee

K. * Budget Justification

Indirect Cost Type

Funds Requested ($)

Funds Requested ($)

* Funds Requested ($)

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

Total Indirect Costs

(Only attach one file.)

(Agency Name, POC Name, and POC Phone Number)

1252-Budget.pdf

Animal costs

Compound resupply

F&A 40.00

RESEARCH & RELATED BUDGET - SECTION F-K, BUDGET PERIOD 2

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2015 06/30/2016

Arietis Corporation

Budget Period 2

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Detailed Budget - Year 2 Page 27

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
* Last Name

RESEARCH & RELATED BUDGET - SECTION A & B, BUDGET PERIOD 3

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* Project Role Base Salary ($)

* Fringe 

Benefits ($) * Funds Requested ($)

9.

8.

7.

6.

5.

4.

3.

2.

1.

Total Funds requested for all Senior Key Persons in the attached file

Total Senior/Key Person

Additional Senior Key Persons:

B. Other Personnel

A. Senior/Key Person

* Number of 

Personnel * Project Role

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

Prefix * First Name Middle Name Suffix

* Fringe 

Benefits ($) * Funds Requested ($)

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

RESEARCH & RELATED Budget {A-B} (Funds Requested)

* ORGANIZATIONAL DUNS:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Project Subaward/Consortium

3

Delete Entry

Dr. Kenneth Coleman PD/PI

3 Senior Scientists 36.00

Arietis Corporation

07/01/2016 06/30/2017 Budget Period 

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Detailed Budget - Year 3 Page 28

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment item * Funds Requested ($)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11. Total funds requested for all equipment listed in the attached file

D. Travel

1. Domestic Travel Costs ( Incl. Canada, Mexico and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

Total Equipment

E. Participant/Trainee Support Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

Number of Participants/Trainees Total Participant/Trainee Support Costs

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {C-E} (Funds Requested)

RESEARCH & RELATED BUDGET - SECTION C, D, & E, BUDGET PERIOD 3

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

Additional Equipment:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2016 06/30/2017

Arietis Corporation

Add Attachment Delete Attachment View Attachment

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Detailed Budget - Year 3 Page 29

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
F. Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

8.

9.

10.

Total Other Direct Costs

G. Direct Costs

Total Direct Costs (A thru F)

H. Indirect Costs Indirect Cost 

Rate (%)

Indirect Cost 

Base ($)

1.

2.

3.

4.

Cognizant Federal Agency

I. Total Direct and Indirect Costs

Total Direct and Indirect Institutional Costs (G + H)

J. Fee

K. * Budget Justification

Indirect Cost Type

Funds Requested ($)

Funds Requested ($)

* Funds Requested ($)

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

Total Indirect Costs

(Only attach one file.)

(Agency Name, POC Name, and POC Phone Number)

1252-Budget.pdf

animal supplies

compound resupply

F&A 40.00

RESEARCH & RELATED BUDGET - SECTION F-K, BUDGET PERIOD 3

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2016 06/30/2017

Arietis Corporation

Budget Period 3

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Detailed Budget - Year 3 Page 30

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
* Last Name

RESEARCH & RELATED BUDGET - SECTION A & B, BUDGET PERIOD 4

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* Project Role Base Salary ($)

* Fringe 

Benefits ($) * Funds Requested ($)

9.

8.

7.

6.

5.

4.

3.

2.

1.

Total Funds requested for all Senior Key Persons in the attached file

Total Senior/Key Person

Additional Senior Key Persons:

B. Other Personnel

A. Senior/Key Person

* Number of 

Personnel * Project Role

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

Post Doctoral Associates

Graduate Students

Undergraduate Students

Secretarial/Clerical

Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

Prefix * First Name Middle Name Suffix

* Fringe 

Benefits ($) * Funds Requested ($)

Acad. 

Months

Sum. 

Months

* Requested 

Salary ($)

Cal. 

Months

RESEARCH & RELATED Budget {A-B} (Funds Requested)

* ORGANIZATIONAL DUNS:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Project Subaward/Consortium

2

Delete Entry

Dr. Kenneth Coleman PD/PI

2 Senior Scientist 24.00

Arietis Corporation

07/01/2017 06/30/2018 Budget Period 

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Detailed Budget - Year 4 Page 31

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment item * Funds Requested ($)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11. Total funds requested for all equipment listed in the attached file

D. Travel

1. Domestic Travel Costs ( Incl. Canada, Mexico and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

Total Equipment

E. Participant/Trainee Support Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

Number of Participants/Trainees Total Participant/Trainee Support Costs

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {C-E} (Funds Requested)

RESEARCH & RELATED BUDGET - SECTION C, D, & E, BUDGET PERIOD 4

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

Additional Equipment:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2017 06/30/2018

Arietis Corporation

Add Attachment Delete Attachment View Attachment

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Detailed Budget - Year 4 Page 32

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
F. Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

8.

9.

10.

Total Other Direct Costs

G. Direct Costs

Total Direct Costs (A thru F)

H. Indirect Costs Indirect Cost 

Rate (%)

Indirect Cost 

Base ($)

1.

2.

3.

4.

Cognizant Federal Agency

I. Total Direct and Indirect Costs

Total Direct and Indirect Institutional Costs (G + H)

J. Fee

K. * Budget Justification

Indirect Cost Type

Funds Requested ($)

Funds Requested ($)

* Funds Requested ($)

Funds Requested ($)

Funds Requested ($)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

Total Indirect Costs

(Only attach one file.)

(Agency Name, POC Name, and POC Phone Number)

1252-Budget.pdf

Animal studies

Outsourced preclinical studies

F&A 40.00

RESEARCH & RELATED BUDGET - SECTION F-K, BUDGET PERIOD 4

* Budget Type:

Enter name of Organization:

* Start Date: * End Date:

* ORGANIZATIONAL DUNS:

Project Subaward/Consortium

07/01/2017 06/30/2018

Arietis Corporation

Budget Period 4

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Detailed Budget - Year 4 Page 33

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Budget Justification

The project will include extensive in vitro and in vivo validation of ADEP4 in combination with a number of other 

antibiotics. A team of 6 Arietis scientists and two consultants will support the project.

Dr. Ken Coleman, PI, Arietis, (10% Y1, 20% Y2-4), is an expert in antimicrobial drug discovery and 

development with 35 years of experience in the Industry. He will be responsible for the overall supervision and 

administration of the project, coordinating interactions with the consultants, the CRO’s and members of the 

team. He will be responsible for research summary reports, and will write the patent filings and papers that will 

result from this project.

Dr. Katya Gavrish, Senior Scientist, (25% Y1, 100% Y2-4), is a specialist in drug discovery and drug 

resistance. Dr. Gavrish contributed to generating the preliminary data that was included in the application. Dr. 

Gavrish will supervise the Research Associates on a day-to-day basis. She will assist as needed with 

individual experiments and data analysis. Dr. Gavrish will also assist Dr. Coleman in writing research reports, 

patent filings and papers that will results from this project.

Dr. Ida Lister, Senior Scientist, (100% Y2-4), is a microbiologist and biochemist. She specializes in 

mechanisms of bacterial drug resistance and will be responsible for performing the PK and efficacy studies. Dr. 

Lister received her PhD from Cambridge University, UK and did postdoctoral research fellowships at Harvard 

and Tufts University.

To be hired, Senior Scientist, (100% Y2-3), an experienced PhD level pharmacologist with project 

management experience will be hired to support the project. This scientist will be primarily responsible for in vivo 

PK/PD data analysis and modelling. This scientist will also work with the business development team and PI at 

Arietis to integrate the data generated from the project into product development plans and target product 

profiles. 

Binu Shrestha, M.Sc. Research Associate, (100% Y1), will perform the hollow-fiber studies in year 1. Binu 

has a Master’s Degree in Molecular Biology from Tufts University and she has worked with Dr. Coleman and

Dr. Gavrish on the project since its inception. 

David Charnuska, B.S., Research Associate, (65% Y1), will perform the in vitro susceptibility and biofilm 

assays in year 1. David will also support the hollow-fiber experiments.

Materials and Supplies

Funds of ~$20,000 for each full time equivalent each year are requested to the purchase of growth media, 

reagents, disposable plastics, and small equipment items such as automatic pipettes to support the work of the 

Arietis team.

Additional funds of $20,000 are requested in year 1, to support the hollow fiber studies.

Additional funds of $107,000 over years 2 and 3 is requested for synthesis of ADEP4 (100 grams), which will 

be sufficient to carry out the proposed studies. Synthesis will be performed (or a comparable CRO), 

who has already optimized a scalable synthetic route and produced the ADEP4 that was used in preliminary 

studies (see quote).

Animal Studies

$44,000 per year (Y2-4) is requested for animal studies to be performed by Arietis at Boston University Medical 

Center. These costs include animal procurement, housing fees, and supplies associated with PK and efficacy 

studies. Additional costs for analytical blood quantification and histopathology to confirm efficacy results are

included.

Additional costs of are requested in year 4 to support the preclinical studies to be performed at

Budget Justification Page 34

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Consultants:

Dr. Steven Leonard ( , 40 hr/year, Y2-4), is a Clinical Pharmacist of Infectious Diseases at Brigham 

and Women’s Hospital and an Assistant Professor of Pharmacy at Northeastern University. Dr. Leonard will 

advise us in all areas of research, especially hollow-fiber resistance studies, and in vivo PK, PD and efficacy.

Dr. Kim Lewis ( , 40 hr/year, Y2-4), is a University Distinguished Professor and the Director of the 

Antimicrobial Discovery Center at Northeastern University. Dr. Lewis is an expert in drug discovery and 

antimicrobial tolerance. Dr. Lewis will advise us in all areas of research throughout this project, especially 

guiding the in vitro and in vivo biofilm studies.

Indirect Costs

Arietis is requesting F&A costs at 40% and fringe benefits at 35%.

Travel

$5000 per year (Y2-4) is requested to support travel of the Arietis team to present work at research 

conferences such as the ASM International Conference on Antimicrobial Agents and Chemotherapy.

Publication

$1000 per year (Y2-4) is requested for publication costs for journals such as Antimicrobial Agents and

Chemotherapy.

 Fee 

Arietis request a fee of 7%.

Budget Justification Page 35

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Section A, Senior/Key Person

Section C, Equipment

RESEARCH & RELATED BUDGET - Cumulative Budget

Section D, Travel

Domestic

Section E, Participant/Trainee Support Costs

Foreign

Tuition/Fees/Health Insurance

Stipends

Travel

Subsistence

Other

Number of Participants/Trainees

1.

2.

3.

4.

5.

6.

1.

2.

Section F, Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

8.

9.

10.

Totals ($)

Total Number Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

Other 1

Other 2

Other 3

Section B, Other Personnel

Section J, Fee

Section I, Total Direct and Indirect Costs (G + H)

Section H, Indirect Costs

Section G, Direct Costs (A thru F)

11

	


 		

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Cumulative Budget Page 44

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
SBIR/STTR Information OMB Number: 4040-0001 

Expiration Date: 6/30/2016

* Program Type (select only one)

* SBIR/STTR Type (select only one)

* 1a. Do you certify that at the time of award your organization will meet the eligibility criteria for a small business as defined in the funding 

opportunity announcement?

* 2. Does this application include subcontracts with Federal laboratories or any other Federal Government agencies?

* If yes, insert the names of the Federal laboratories/agencies:

* 3. Are you located in a HUBZone? To find out if your business is in a HUBZone, use the mapping utility provided by the Small Business 

* 4. Will all research and development on the project be performed in its entirety in the United States?

* Explanation:

* 5. Has the applicant and/or Program Director/Principal Investigator submitted proposals for essentially equivalent work under other 

Federal program solicitations or received other Federal awards for essentially equivalent work? 

* If yes, insert the names of the other Federal agencies:

* 6. Disclosure Permission Statement: If this application does not result in an award, is the Government permitted to disclose the title of 

your proposed project, and the name, address, telephone number and e-mail address of the official signing for the applicant organization, 

to organizations that may be interested in contacting you for further information (e.g., possible collaborations, investment)?

* 7. Commercialization Plan: If you are submitting a Phase II or Phase I/Phase II Fast-Track Application, include a 

Commercialization Plan in accordance with the agency announcement and/or agency-specific instructions.

* Attach File:

Questions 1-7 must be completed by all SBIR and STTR Applicants:

If no, provide an explanation in an attached file.

Administration at its web site: http://www.sba.gov 

SBIR STTR

Both (See agency-specific instructions to determine whether a particular agency allows a single submission for both SBIR and STTR)

Phase I Phase II

Fast-Track (See agency-specific instructions to determine whether a particular agency participates in Fast-Track)

Yes

No

9

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Add Attachment Delete Attachment View Attachment

1250-CommercializationPlan.pdf Add Attachment Delete Attachment View Attachment

* 1b. Anticipated Number of personnel to be employed at your organization at the time of award.

	


 		

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SBIR/STTR Information Page 45

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
* 8. Have you received SBIR Phase II awards from the Federal Government? If yes, provide a company commercialization history in 

accordance with agency-specific instructions using this attachment. 

* Attach File:

* 9. Will the Project Director/Principal Investigator have his/her primary employment with the small business at the time of award?

* 10. Please indicate whether the answer to BOTH of the following questions is TRUE:

(1) Does the Project Director/Principal Investigator have a formal appointment or commitment either with the small business directly 

(as an employee or a contractor) OR as an employee of the Research Institution, which in turn has made a commitment to the 

small business through the STTR application process; AND 

(2) Will the Project Director/Principal Investigator devote at least 10% effort to the proposed project?

* 11. In the joint research and development proposed in this project, does the small business perform at least 40% of the work and the research 

institution named in the application perform at least 30% of the work?

SBIR-Specific Questions:

STTR-Specific Questions:

SBIR/STTR Information

Questions 8 and 9 apply only to SBIR applications. If you are submitting ONLY an STTR application, leave questions 8 and 9 blank and proceed to 

question 10.

Questions 10 and 11 apply only to STTR applications. If you are submitting ONLY an SBIR application, leave questions 10 and 11 blank.

1251-Company Commercialization Add Attachment Delete Attachment View Attachment

Yes

No

Yes

No

Yes

No

Yes

No

	


 		

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SBIR/STTR Information Page 46

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Commercialization Plan 

Value of the SBIR Project, Expected Outcomes, and Impact. 

 

The goal of this SBIR Fast-Track project is to develop a combination therapeutic consisting of ADEP4 and an 

approved antibiotic. This combination is for the treatment of refractory and recurrent S. aureus infections, 

including Methicillin Resistant S. aureus (MRSA). The FDA had published extensive Guidance for Industry on 

Developing Drugs for Treatment for Acute Bacterial Skin and Skin Structure Infections and we will use these 

guidelines throughout our development program. An important characteristic of our therapeutic is the ability to 

act against biofilms, stationary phase, persister cells and complicated forms of infection. The therapeutic 

should result in eradication rather than mere suppression of infection, which is critical for clearance of biofilm 

infected medical devices and in immunocompromised patients. This project constitutes a Phase 1 feasibility 

study, which will enable us to determine whether the resistance rate of the combined therapeutic is low enough 

to warrant further investigation. In Phase 2, the combination will be tested in animal models of MRSA infection, 

which are largely predictive of outcomes in humans. We have purposefully chosen models in which standard 

front-line therapies fail, allowing our therapeutic combination to demonstrate clear superiority. Phase 2 

constitutes a series of standard preclinical development studies, which will enable a pre-IND meeting with the 

FDA upon this project’s completion. 

Staphyloccoccus aureus, was discovered in the 1880s and skin and soft tissue conditions such as boils were 

subsequently associated with the bacteria. S. aureus was also known to progress to bacterial pneumonia and 

septicemia, which were typically fatal prior to the age of antibiotics. Since the 1940’s, antibiotic treatment of S. 

aureus infection was routine, however resistance to resistance to penicillin developed almost immediately. 

Similarly, the first case of methicillin resistance in a patient was documented in 1961 and the first case reported 

in the US was in 1968. MRSA refers to resistant to the entire class of beta-lactams, which includes the 

penicillins and cephalosporins. S. aureus continues to evolve resistance at a rapid pace. Vancomycin 

resistance has been documented since 2002 and it is not a matter of if resistance will develop, but when. 

Thus, S. aureus has evolved from a controllable nuisance into a major problem, within a generation. S. aureus

is ubiquitous - most people carry the bacteria on their skin and it does not typically cause an active infection. 

This is even true for carriers of MRSA. MRSA is often categorized according to where the infection was 

acquired: hospital-acquired MRSA (HA-MRSA) or community-associated MRSA (CA-MRSA). HA-MRSA has 

increased during the past decade and it is typically among the most frequent of all hospital acquired infections. 

CA-MRSA is caused by newly emerging strains and can infect healthy people with no warning. CA-MRSA may 

begin as skin or soft tissue infections, but can progress into a life-threatening infection. CA-MRSA is also 

occurring with increasing frequency and people who are in close physical contact, such as athletes and 

soldiers, are particularly susceptible. USA300 is a common strain of CA-MRSA that is responsible for rapidly 

progressive, fatal diseases including necrotizing pneumonia, severe sepsis and necrotizing fasciitis. 

S. aureus is responsible for ~1,200,000 hospital infections each year and there are an estimated 94,000 lifethreatening infections from MRSA. These represent 20% of all hospital infections. According to the Journal of 

the American Medical Association, MRSA is responsible for an estimated 18,650 deaths in the US. The CDC 

estimated 10,800 deaths occurred in the U.S. in 2005 and over half of these were linked to MRSA. The rate of 

MRSA infections recorded at U.S. academic hospitals doubled between 2003 and 2008 (David et al., 2012). 

Community acquired MRSA is estimated to have a $1.4-13.8 billion on society, while the US jail system and 

Army may be experiencing annual total costs of $7-11 million ($6-10 million direct medical costs) and $15-36 

million ($14-32 million direct costs), respectively (Lee et al., 2013). Surprisingly, for every death from MRSA, it 

is estimated we spend only $570 on research. This is a small amount compared to spending for AIDS, for 

which $69,000 is spent for every AIDS death, even though MRSA deaths far outnumber those from AIDS. 

S. aureus biofilm infections such as endocarditis, osteomyelitis or those on implanted medical devices, are 

essential untreatable. For example, according to the Infectious Disease Society of America, a minimum 8-

week course is recommended, although some experts suggest an additional 1-3 months and possibly longer 

for chronic infection or if debridement is not performed (Liu et al., 2011). We found that a stationary population 

of S. aureus is completely refractory to killing by traditional antibiotics. This fact widely appreciated by 

researchers or clinicians. The refractory nature of stationary cells can be explained in the terms of target 

Commercialization Plan Page 47

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
availability - many of the cellular targets of traditional antibiotics are not active in stationary phase. Indeed, 

bactericidal antibiotics require an active target to be corrupted in order to produce killing. Thus, 

aminoglycosides cause mistranslation, which creates toxic misfolded proteins; fluoroquinolones convert DNA 

gyrase/topoisomerase into an endonuclease which cleaves DNA; and β-lactams activate autolysins that 

destroy the cell wall. 

We reasoned that cells exhibiting drug tolerance could be killed by an antibiotic which activates and corrupts a 

cellular target. Remarkably, the antimicrobial acyldepsipeptide (ADEP), functions by precisely this mechanism. 

ADEP causes dysregulation of the ClpP protease, relieving it from regulatory control and the requirement to 

use ATP. ADEP activates proteolysis in stationary phase, causing even dormant cells to self-digest. 

Interestingly, proteolysis is responsible for the shutdown of many of the other cellular targets of antibiotics 

during dormancy and stationary phase. If these targets are not properly shut down, traditional antibiotics can 

be effective. This is a major conceptual advance that will allow us to treat incurable infection for the first time. 

In support of our rational, we found that the dysregulation of proteolysis by ADEP caused exceptional killing of 

biofilms and dormant cells. ADEP also caused increased susceptibility to killing by other antibiotics, since the 

traditional antibacterial targets were not properly inactivated by proteolysis. Combining ADEP4 with antibiotics 

rifampicin or linezolid produced complete sterilization of stationary and biofilm S. aureus cultures. Importantly, 

ADEP4 in combination with rifampicin sterilized MRSA in a mouse model of deep-seated infection, where 

conventional antibiotics had very little effect. 

ADEP does have a liability – ClpP is not essential, and a high-frequency of null mutations leads to resistance. 

However, knowing the potential of ADEP to target dormant cells and render cells to be more sensitive to killing 

by antibiotics, it makes sense to combine it with conventional antibiotics, which will also solve the resistance 

problem. In Phase I of this project, we will identify the best ADEP partnering antibiotic and we will evaluate the 

combinations for the potential to develop resistance in the hollow-fiber model. The frequency of resistance 

development will be the go-no-go decision point for this project. We will not enter into Phase 2 until we 

conclusively demonstrate that resistance development for the combined therapeutic will not be a concern. 

Typically, a resistance rate higher than 10-8 is considered too high to enter development for an antimicrobial. 

The approach of combining antibiotics to overcome resistance is not new. In S. aureus, spontaneous 

resistance mutants arise at a frequency of around 10−8 for selections with rifampicin or fusidic acids, but are 

undetectable (frequency <10−11) for a combined treatment. Because an infection may contain more than 108

cells, rifampicin is not used alone for the treatment of S. aureus, but only in combination with another agent. 

Since ADEP4 has a unique target and mechanism of action, and an amazing potential to target drug tolerant 

cells, it makes sense to combine it with another antimicrobial. In this project we will test combinations of 

ADEP4 and other antibiotics for activity against stationary phase, biofilms and difficult to treat infection models, 

to determine the best partnering antibiotic. 

Because resistance to MRSA is a major problem, it continues to be the focus of new product launches, 

underscoring both an unmet medical need and a clear commercial potential. In particular, there are two 

lipoglycopeptides under development, dalbavancin and oritavancin, and one oxazolidinone, tedizolid. Based 

on their mechanisms, it is unlikely these drugs will have activity against biofilms or stationary cells, or would be 

effective in a model of deep-seated infection. However, these agents could make suitable partners for ADEP4, 

once they are available. Clearly the continued activity in this arena, and the commercial success of variants of 

existing antibiotics, highlights a market hungry for meaningful improvements over currently available drug 

products. Recurrence is most likely due to the survival of drug-tolerant persister cells that repopulate the 

infection once the drug concentration drops. The ability of our combination therapeutic to kill persisters, is likely 

to reduce recurrence and satisfy this unmet medical need. 

All of the considerations outlined above also apply not only to skin and soft tissue infections, but to other 

infections caused by MRSA, such as osteomyelitis, endocarditis, and biofilm infected joints and catheters. The 

current project is expected to result in multiple new drug products such as topical agent to eradicate nasal 

carriage, creams or ointments for topical use, combinations with systemic agents for systemic use, catheter 

lock solutions and medical device coatings. A product for treatment of skin and soft tissue infections is the 

primary objective of this development plan. Other commercial product paths are envisioned as future market 

expansions. 

Commercialization Plan Page 48

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Key technology objectives. 

The ultimate goal of the project will be preparation of a development candidate suitable for entry into focused 

preclinical drug development with input from the FDA in order to support Investigational New Drug (IND)-

enabling studies. A summary of key technology objectives relating to the milestones follows: 

Aim 1. Identify the best sterilizing combination of ADEP4 and an approved antibiotic. ADEP4 will be paired with 

known antibiotics such as daptomycin, linezolid, ciprofloxacin, gentamicin, and oxacillin, and examined for its 

ability to sterilize stationary and biofilm populations of S. aureus. ADEP/antibiotic combinations will also be 

tested for maximum killing and low resistance development using a hollow-fiber model of in vitro infection. 

Milestone: Advance 2 ADEP4/antibiotic combinations to in vivo studies. Combinations which effectively sterilize 

biofilms and stationary populations of S. aureus, and have a rate of resistance development <10-9 in the hollow 

fiber model will be advanced. 

Aim 2. In vivo testing of sterilizing combinations. PK parameters and initial efficacy of two ADEP4/antibiotic 

combinations will be performed in mice. The PK/PD driver of ADEP efficacy will be determined using the 

mouse model of deep-seated MRSA infection. Efficacy of each combination will be tested in an implanted 

tissue cage biofilm model. Milestone: Determine the PK of the combined agents, the pharmacodynamic driver 

of efficacy and treat a previously incurable model of in vivo biofilm MRSA infection. 

Aim 3. Preclinical development. Acute, dose-range finding, and 14 day repeated dose toxicity studies will be 

performed in rats. PK studies in rats will provide the basis for selecting doses to be tested in humans. 

Ames and chromosomal aberration genotoxicity studies will also be performed. 

Taken together, these studies will form the basis for a risk-benefit assessment prior to the pre-IND meeting 

with the FDA and entry into human clinical trials. We will work with the FDA to define the remaining preclinical 

steps if not already covered. 

 In house studies in the mouse model to determine the PK/PD driver and magnitude of each drug, will 

help with human dose prediction. 

 Preliminary toxicology and local irritation assessments in preparation for selection of the IND 

combination and dose selection to proceed into IND-enabling studies. 

 Prior to the final design of the definitive GLP toxicology and safety program, a Medical Advisory Board 

will be convened to aid in the design of the clinical program to ensure that the preclinical program 

submitted with the IND properly supports the intended development path. 

 Dose range-finding studies along with metabolite/CYP profiling and plasma protein-biding assays will 

be performed in house. 

 

 GLP and preclinical studies, which will be outsourced to will include genotoxicity, bioanalytical 

method development and validation in 2 species, definitive Toxicology studies in 2 species, and singledose PK/ADME studies in 2 species. 

 Safety pharmacology studies, to include CNS and respiratory in the rat and , 

will also be outsourced 

 

 Formulation development and validation, GMP production, stability of both drug substance and drug 

product will be outsourced to specialists in GMP production of 

clinical trial materials. 

 Oversight of all outsourced GLP and GMP work will be provided by Ken Coleman who has 35 years of

experience designing development programs and providing these capabilities in both large 

pharmaceutical and small biotech companies. 

Commercialization Plan Page 49

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Success in these milestones will enable Arietis to enter Phase III of the project, focused drug development, 

ultimately leading to commercial products – a novel skin and soft tissue treatment capable of overcoming 

complicated MRSA infection. The ultimate product profile will be determined by data obtained during research 

and development efforts, in conjunction with the clinical input of a Medical Advisory Board (MAB) composed of 

key opinion leaders in the field of infectious disease. Specific clinical input will be gathered on the best ways to 

use the drug combination based on its inherent pharmacological characteristics, and how best it can be used 

as an addition to the existing armamentarium. 

 The FDA has provided an excellent guidance to inform our development path in its “Guidance for 

Industry on Developing Drugs for Treatment for Acute Bacterial Skin and Skin Structure Infections” and 

“Guidance for Industry and FDA Staff: Early Development Considerations for Innovative Combination 

Products”. The FDA has also recently released “Draft Guidance for Industry on Antibacterial Therapies for 

Patients With Unmet Medical Need for the Treatment of Serious Bacterial Diseases.” The guidance presents 

streamlined approaches to evaluating new antibacterials for patients with unmet medical needs, and for 

pathogen-focused antibacterials. We expect our combination therapeutic to fall under Tier C, which could 

significantly streamline both the cost of clinical trials and the time to market. Pathogen-focused MRSA 

treatments addressing unmet medical need would fall in between, in tiers B and C. Approval would rely heavily 

on human pharmacokinetic (PK) data combined with preclinical data in animals. Tier B drugs would include 

broad-spectrum antimicrobials that also act against a multidrug-resistant pathogen, such as multidrugresistant Enterobacteriaceae. Tier C would include antimicrobials with very narrow multidrug-resistant 

pathogen coverage, such as an antibiotic that is effective only against Acinetobacter or one that targets 

only Pseudomonas aeruginosa. Deciding whether a drug should be assigned to tier B versus tier C would 

depend on the feasibility of studies, the degree of unmet medical need, and the strength of preclinical data, 

based on conversations with the FDA. Coupled with the clinical guidance of the MAB this input will be used in 

outlining our clinical program design, which will in turn guide the detailed definition of the preclinical program 

needed to support it. Arietis management has extensive experience in establishing and coordinating the input 

from MABs, Regulatory Affairs, and designing clinical and preclinical programs, as well as clinical study design, 

implementation, and reporting. 

In addition to our in house capabilities, several contract research organizations (CROs) are available with the 

expertise to conduct preclinical studies compliant with current good laboratory practices (GLPs) for inclusion in 

an IND. Arietis management has extensive experience in choosing and managing high quality CROs for this 

purpose. Successful completion of Phase II is therefore an integral part of our research and development plan, 

aimed at commercializing a new, more effective MRSA therapy. 

Scientific benefits and the non-commercial impacts of the project. In the course of Phase I and transition to 

Phase II work, Arietis has identified scientific questions relating to the benefit of an antimicrobial, which targets 

drug tolerance and functions through an activation mechanism. Conceptually, one can image numerous added 

benefits conferred by eradicating an infection as opposed to suppressing it. The clinical benefits of eradicating 

an infection may especially pertain to deep-seated and complicated infections which are refractory to current 

treatments. Now, for the first time we have the tools to answer some fundamental questions: For example, will 

targeting drug tolerance cell populations lead to shorter durations of therapy and less frequent emergence of 

resistance? Is recurrent infection caused by pathogen re-colonization or drug tolerance? Indeed, we are 

excited about the possibility of uncovering a link between difficult to treat infection, drug tolerance, classical 

resistance and biofilms. New findings in this area may help shift the paradigm of how we think about drug 

tolerance and drug resistance. 

Company, Production and Marketing Plan, Finance Plan, and Revenue Stream 

Arietis Corporation was founded by Dr. Kim Lewis to commercialize his method for eliminating persisters, and 

thereby identify or create agents that sterilize, rather than simply suppress, infections. In May, 2008 the 

Company was awarded an SBIR Phase I grant, which was successful in establishing compound library 

screens and identifying validated hits in vitro for further evaluation in vivo as potential drug development 

candidates. In November, 2008 the company moved into its present location at 650 Albany Street in Boston, 

Massachusetts, a mecca of innovation in the biotech and pharma industries. The Company currently occupies 

1400 sq. ft. of laboratory and office space, with additional access to animal facilities at Boston University 

School of Medicine. The laboratory space is allocated to biology. The current space will be sufficient for us to 

Commercialization Plan Page 50

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
perform the planned work, supplemented as necessary by collaborations with outside labs for the conduct of 

some specialized animal studies and synthetic chemistry scale up. 

Dr. Kenneth Coleman serves as PI. Dr. Coleman has a long history of successful antimicrobial drug discovery 

and development, both in major global pharma companies and small start-up environments. Daily scientific 

activities are overseen by Michael LaFleur, Chief Operating Officer, one of the inventors of the company’s core 

technologies. His team includes both PhD-level scientists and a veterinarian. We anticipate hiring one

additional employee, to serve as senior scientist and project manager, analyzing and model PK/PD data 

generated during the project. Thus a team of 6 people will be working on the project, along with extramural 

expertise in the areas of pharmacology, chemistry and pre-clinical animal studies. Dr. Coleman will also 

coordinate the input of Drs. Leonard, Lewis and LaFleur as well as members of the SAB. Dr. Coleman will 

manage outsourcing of work conducted off-site under this program. 

The core competence of our team is in anti-infective drug discovery and preclinical and clinical drug 

development, with emphasis on antiinfectives and especially activity against persister cells. Our expertise is 

supplemented by a Scientific Advisory Board made up of outstanding scientists: 

In addition to these key experts, the Company benefits from the advice of its founder, Dr. Kim 

Lewis, Professor of Biology at Northeastern University. 

As we move on to Phase III and beyond, the company will undergo substantial expansion as required to 

transition from a research to a drug development focus. It should be noted that creation of drug development 

capabilities is something that Coleman has accomplished for many of his previous employers. 

The Team 

● Dr. Kenneth Coleman, PI, has a number of successful drugs in his portfolio, including clavulanic acid and 

gemifloxacin. Dr. Coleman has more than 35 years of experience in antimicrobial drug discovery and 

development in both large pharma and small biotech companies. 

● Dr. Michael LaFleur, Chief Operating Officer, was responsible for coordinating the work described in 

Preliminary Studies. He serves as the project manager and grant administrator. 

● Dr. Kim Lewis, Consultant, is Director, Antimicrobial Discovery Center of NEU, and Founder of Arietis. Dr. 

Lewis is an expert in antimicrobial drug resistance, drug tolerance, and anti-infective drug discovery. 

● Dr. Steven Leonard, Consultant, is a Clinical Pharmacist of Infectious Diseases at Brigham and Women’s 

Hospital and an Assistant Professor of Pharmacy at Northeastern University. Dr. Leonard will contribute all 

areas of research, especially hollow-fiber resistance studies, and in vivo PK, PD and efficacy. 

● Dr. Ida Lister, Senior Scientist is a microbiologist and biochemist. She specializes in mechanisms of 

bacterial drug resistance. Dr. Lister received her PhD from Cambridge University, UK and did postdoctoral 

research fellowships at Harvard and Tufts University. 

● Dr. Ekaterina Gavrish, Senior Scientist, is a microbiologist who worked with Dr. Lewis for many years. She 

is a specialist in drug discovery and drug resistance. 

● Ms. Binu Shrestha, is a veterinarian who also holds a Master’s Degree in Molecular Biology from Tufts 

University. 

Several outstanding scientists are members of the Scientific Advisory Board of Arietis. In addition to Drs. 

Lewis: 

● 

Commercialization Plan Page 51

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
 

● 

● 

. 

● 

. 

Funding history. The company has been supported by SBIR funds in the past. 

NIH/NIAID 5R43AI098327-02 

Compounds to treat Helicobacter pylori infection. 

06/15/2012-05/31/2014. 

Establish proof of concept for developing novel small molecules capable of eradicating H. pylori infection 

NIH/NIAID 1R44AI102452-01 

Narrow-spectrum Agents Acting against Helicobacter pylori 

07/01/2012-06/30/2013 

This successful Phase I project was continued in a Phase II grant 

NIH/NIAID 4R44AI102452 - 02

Narrow-spectrum Agents Acting against Helicobacter pylori 

07/01/2013-06/30/2016 

Iterative medicinal chemistry optimization will be combined with detailed validation of each series in 

Phase II. 

NIH/NIAID 1R43AI074258-01A1 

Therapy against Recalcitrant C. albicans Infection 

05/15/2008 – 05/14/2010. 

This successful Phase I project was continued in a Phase II grant: 

NIH/NIAID 5R44AI074258-04. 

Therapy against Recalcitrant C. albicans Infection 

07/01/2010 – 12/30/2013

This project seeks to develop topical therapy for recurrent vulvovaginal candidiasis. 

NIH/NIDCR 5R43DE020880-02 

2-adamantanamine based therapeutic for recurrent oropharyngeal Candidiasis. 

05/01/2010 – 04/30/2012. This project seeks to develop therapy for oral thrush. 

Corporate vision – from small R&D business to a commercial entity. 

Arietis was founded on a basic scientific breakthrough: a means to eliminate the persister cells that are 

responsible for recurrent and recalcitrant fungal infections. The Company has successfully leveraged this 

invention into a drug discovery engine, with tangible results warranting further exploration and development 

into safe and effective therapies made available to patients through commercialization. 

Commercialization Plan Page 52

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
As drug development proceeds through its lifecycle, development costs increase along with probabilities of 

success. The key to success in very early stage endeavors is to very carefully match funds available with 

strategically chosen experimentation that maximizes the chances of proceeding to the next stage of 

development. 

Next steps in commercialization include identifying ways to defray or defer costs while simultaneously reducing 

program risk. This can best be done by visualizing what the end commercial product will look like and who will 

be using it (both the patient and prescriber). The result is a product profile that can be used to drive the 

roadmap of further development. We plan to take advantage of the NIH’s Niche Assessment program during 

our Phase I progress, with the result that we have had access to Key Opinion Leaders and selected end user 

(prescriber) advisory input in defining our product profile, as well as initial contacts with targeted pharma 

companies expressing an interest in learning more about this project and our lead candidate as accumulate 

additional data through the conduct of this Phase II program. 

Focusing on the primary (initial) commercial profile – in this case skin and soft tissue infections caused by 

MRSA -- we can identify the most likely regulatory requirements for marketing approval by the FDA, and 

design an appropriate and efficient clinical trial program strategy for each commercial application. For 

example, the most likely initial approval will be for second line treatment after failure with a current standard 

treatment, linezolid. With an emphasis on effecting a microbial cure in addition to a clinical cure and 

decreasing frequency of recurrence, this product also has the potential to move into a first line position in the 

future for immunocompromised patient populations most affected by issues of recurrence. 

With a clinical trial program outlined we will then design an appropriate and efficient preclinical program to 

support entry into clinical trials. Outlining a preclinical program allows us to identify an efficient late research 

plan to position us for the timeliest and most cost-effective entry into focused drug development with the most 

worthwhile drug candidate or candidates. 

Once entering focused development, the costs and risks are well understood from a historical perspective. At 

this time, during initial preclinical development, we anticipate having a sufficiently robust dataset and 

understanding of likely development pathways and regulatory requirements to begin entering into discussions 

with potential industry partners and investors, to further the goals of the company. While we will continue to be 

open to both types of investment, as well as continued research support through programs such as SBIR, it is 

most likely that we would seek an industry partner for our first and most advanced commercial product. The 

reasons for this are 2-fold: from an industry perspective, a proven program from a small R&D entity can 

compete favorably with unproven internal research programs, providing faster and more cost-effective ways to 

bring a product to market; and from Arietis’ perspective, the existence of a mature, established marketing and 

distribution capability within the corporate partner provides the fastest and most efficient means to get safe and 

effective therapies into the hands of the physicians who will prescribe them and the patients whose lives will 

benefit from them. Through the NIH’s Niche Assessment process we will become familiar with the potential 

partners most interested in licensing this product, and have initiated discussions anticipated to progress as 

more data become available through the studies supported by this application. We have already entered into 

discussions with several large pharmaceutical companies based on our preliminary data and the Nature 

publication. There is a high level of interest however, based on our considerable experience with large Pharma 

and Biotech, it may be unrealistic to obtain a meaningful commitment from a partner at this early stage of 

development. We plan to partner after we have generated preclinical data in Phase II. 

The revenue stream to be generated through successful partnership and commercialization of this first product 

then make possible both the continued development of other therapies, and the establishment of additional 

capabilities such as manufacturing, marketing and distribution necessary to transform a research and 

development undertaking into a fully integrated pharmaceutical company, with a robust pipeline of drug 

candidates to treat an array of diseases across the anti-infective spectrum. 

Initially and in the absence of a full integration of pharmaceutical company capabilities we will outsource 

required work, especially in order to comply with government regulations (e.g., concerning good manufacturing 

practices, or GMP, for the production of drug substance and drug product; good laboratory practices or GLPs 

for the studies to be included in the IND, and eventually good clinical practices, or GCPs, for the conduct of 

Commercialization Plan Page 53

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
clinical trials to support the marketing application). Work such as basic research, mechanism of action, 

resistance testing, etc., that is not subject to specific regulation we will perform in-house, supplemented as 

necessary or desirable with academic expertise (e.g., novel assays or animal models) through collaboration 

with leaders in the relevant areas. 

Decisions on whether to seek additional private investment, additional partnerships, or access public funds via 

an IPO will be data-driven in the future, based on factors such as the number and stage of additional drug 

candidates, the costs and risks of carrying them forward, and any available revenue streams such as royalties 

or commercial sales. In general, it is our bias to hold both individual programs and company stock as long as 

possible in order to reduce risk and increase value, while still providing the resources necessary to advance 

drug candidates efficiently toward the marketplace. 

Market, Customer, and Competition. 

Market and Customer. 

Despite a global market size of $42 billion for anti-infectives, there is a substantial unmet need for novel 

antimicrobial compounds (Hamad, 2010). Very few novel antimicrobials have reached market in the last 10 

years. However, there are no sterilizing antimicrobials which are capable of eradicating, rather than 

suppressing, an infection. Reflecting the paucity in the discovery of new drugs, even narrow-spectrum 

antibiotics which are novel and not subject to pathogen resistance do very well in the marketplace. The total 

US market for antibiotics is approximately $25 billion, with several narrow-spectrum compounds each earning 

more than $1 billion annually. 

Glycopeptides, such as vancomycin, are the treatment of choice against MRSA. In 2006, sales of 

glycopeptides totaled $680 million. Vancomycin-resistant strains have begun to appear and in such cases 

linezolid and daptomycin are typically used. Worldwide sales of linezolid (Zyvox, Pfizer) exceeded $1B per 

year for the period 2007-2010 and daptomycin (Cubicin, Cubist) generated revenue $600M in the US. The 

market for these agents is expected to grow. 

It is important to note that a combined agent, such as Arietis is developing, does not need to replace current 

therapies in order to gain market share, because it is intended to be added to -- thereby increasing the 

effectiveness of -- existing products. Most importantly, the customer for these new agents will benefits from 

successful treatment without the pitfalls of inadequate infection control and persistent recurrence. 

Skin and soft-tissue infections can become complicated, (cSSTI), defined as patients with deeper soft-tissue 

infections, surgical/traumatic wound infection, major abscesses, cellulitis, and infected ulcers and burns and 

these infections are often difficult to treat. Bactericidal agents are preferred for these deep-seated infections, 

although they are still difficult to treat, often requiring longer courses of therapy and surgical debridement. 

Recurrent SSTI is not considered complicated, but is still a problem and nasal decolonization is recommended 

if optimized wound care and hygiene were followed. Oral therapy is not recommended decolonization and 

should be reserved for active infection unless initial decolonization attempts fail. An oral agent in combination 

with rifampin, may be considered for decolonization, provided the strain is susceptible. No clinical trials have 

evaluated the role of oral antimicrobials for treatment of recurrent CA-MRSA SSTI and even the definition of 

recurrence is not standardized. 

In addition to SSTI, MRSA bacteremia and infective endocarditis are serious diseases associated with high 

morbidity, and mortality rates are 30%– 37% for MRSA endocarditis. Vancomycin is still the mainstay of 

therapy for MRSA bacteremia and endocarditis, but obviously it is not effective in all cases. Daptomycin is an 

alternative to vancomycin for the treatment of MRSA bacteremia or endocarditis. Emergence of reduced 

susceptibility to daptomycin was observed in several daptomycin-treated patients who experienced failure of 

therapy. 

Similarly, high failure rates have been observed in the treatment of MRSA pneumonia, particularly in ventilatorassociated pneumonia (VAP). Failure of vancomycin may be related to poor penetration into the lung 

epithelial. Linezolid is an alternative, while daptomycin bind to lung surfactant, rendering it ineffective. 

Commercialization Plan Page 54

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Osteomyelitis is also very problematic. Surgical debridement of necrotic bone, drainage of abscesses and 

antimicrobial therapy is required. Vancomycin remains the primary treatment for MRSA osteomyelitis and 

failure rates from 35%–46% have been reported. There are no controlled trials of MRSA osteomyelitis, but 

small trials of MSSA osteomyelitis suggested higher cure rates were associated with combination rifampicin 

therapy. The optimal duration of therapy for osteomyelitis is unknown. 

Since we expect our combinational therapeutic to be more effective compared to current agents, our 

therapeutic has the potential to improve cure rates and prevent relapse. The main driver for the current project 

is that a therapeutic that specifically targets drug tolerant cells and is highly cidal to bacteria will improve the 

effectiveness of chemotherapy and decrease relapse. An antibiotic with these performance characteristic is 

therefore anticipated to have a market advantage. 

Expert opinion, collected through consultants and clinicians, noted an urgent need to find novel technologies 

and new classes of antimicrobials within this arena. They also noted that the severe debilitation is frequently 

observed in these patients with osteomyelitis, endocarditis and VAP. Current antibiotic therapies’ inability to 

completely eliminate infection may be a major cause of clinical failure. Based on this input, end-users expect 

that this type of therapy would be used initially as a second line agent for uncomplicated infections with the 

possibility for first line agent using for the complicated infections which we target. 

In another regard, and consistent with our business plan, the customer is one or more pharmaceutical 

companies with currently marketed antibiotic agents that can improve their product offerings, both in terms of 

therapeutic effectiveness and consequent increased market share. We have identified and initiated 

conversations with potential partners, and will continue these discussions as more data on the performance of 

our product candidates become available through the performance of the studies proposed in the current 

application. 

Competition. 

Vancomycin has long been the standard of care for MRSA treatment, but its market share is increasingly being 

replaced with newer agents, such as dapotmycin or ceftobiprole. These and other products currently available 

for treatment of S. aureus are summarized in the Competition Assessment Table below. None of these agents 

has activity against stationary and persister cell populations of S. aureus. There are currently no antibiotics

capable of sterilizing infection, and therefore all products are subject to failure, are ineffective or require longterm regiments. We have not found a strongly competitive pipeline of new classes in development vying for 

FDA approval at this point. A majority of pharmaceutical companies have suspended new research in the gram 

positive area or are developing molecules based on iterative improvements of known agents. In this regard, 

there is no direct competitor for Arietis’ approach, as it represents a first in class novel therapeutic. In addition, 

approved agents that demonstrate activity complementary to ADEP4 will benefit from combination therapy, 

meaning that markets for both are expected to expand, rather than compete with each other. In one sense, the 

market for Arietis’ will not be limited by competition but by the range of currently available agents that provide 

enhanced activity in combination. Determining the scope of this is an active area of investigation. At the same 

time, however, an effective combination can be expected to out-compete any single agent in the treatment of 

recurrent infections, as no single agent can achieve the sterilization necessary to prevent recurrence.

Competition Assessment Table 

Product Name Manufacturer Relevance

Activity 

against 

Persisters

B-lactams multiple newer agents like Ceftobiprole, may be 

effective against MRSA

No

Ceftaroline Forest/AstraZene

ca/Dainippon 

Sumitomo

IV only, cephalosporin, approved for 

complicated skin/soft tissue infections and 

community-acquired pneumonia 

No

Clindamycin multiple bacteriostatic No

Daptomycin Cubist IV only, cidal, membrane acting No

Commercialization Plan Page 55

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Linezolid Pfizer bacteriostatic new drug of last resort No

QuinupristinDalfopristinKing 

Pharmaceuticals

IV only, off label MRSA use, bactericidal No

Rifampicin Sanofi Aventis

cidal, used in combination with other 

antibiotics due to high resistance No

Telavancin Astellas not currently available due to 

manufacturing issues No

Tetracyclines multiple bacteriostatic, resistance a concern No

TMP-SMX Roche, GSK bacteriostatic, off label use for MRSA No

Vancomycin ViroPharma mainstay of MRSA therapy, slow 

bacteriocidal activity and resistance are 

concerns 

No

Dalbavancin Durata 

Therapeutics

IV, lipoglycopeptide under development No

Oritavancin The Medicine Co. IV, lipoglycopeptide under development No

Tedizolid Trius/Cubist oxazolidinone under development No

The trend for the basis of competition in this arena will continue to be best performance as there are numerous 

products available as well as generics. In this case, performance will be measured by ability to treat 

complicated SSTI, osteomyelitis, VAP, bacteremia and endocarditis, where there is clearly a need for 

improvement. 

Intellectual Property (IP) Protection. 

 

 

David MZ, Medvedev S, Hohmann SF, Ewigman B, Daum RS, (2012) Increasing burden of methicillinresistant Staphylococcus aureus hospitalizations at US academic medical centers, 2003-2008. Infect 

Control Hosp Epidemiol.

Hamad, B., (2010) The antibiotics market. Nat. Rev. Drug Discov. 9, 675-676.

Lee, B. Y., A. Singh, M. Z. David, S. M. Bartsch, R. B. Slayton, S. S. Huang, S. M. Zimmer, M. A. Potter, C. M. 

Macal, D. S. Lauderdale, L. G. Miller & R. S. Daum, (2013) The economic burden of communityassociated methicillin-resistant Staphylococcus aureus (CA-MRSA). Clin Microbiol Infect 19: 528-536.

Liu, C., A. Bayer, S. E. Cosgrove, R. S. Daum, S. K. Fridkin, R. J. Gorwitz, S. L. Kaplan, A. W. Karchmer, D. P. 

Levine, B. E. Murray, J. R. M, D. A. Talan & H. F. Chambers, (2011) Clinical practice guidelines by the 

infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus

infections in adults and children: executive summary. Clin Infect Dis 52: 285-292.

Information also collected from the NIH CDC websites. 

Commercialization Plan Page 56

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Company Commercialization History 

Arietis Corporation has not received more than 15 SBIR Phase II awards from the Federal Government during 

the preceding 5 fiscal years. 

Prior SBIR Phase II Awards Page 57

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
PHS 398 Cover Page Supplement

1. Project Director / Principal Investigator (PD/PI)

* Title:

* Street1:

* City:

* Country:

Street2:

County/Parish:

* State:

* Zip / Postal Code:

Prefix: * First Name:

Middle Name:

* Last Name:

Suffix:

2. Human Subjects

Clinical Trial?

* Agency-Defined Phase III Clinical Trial?

* First Name:

Middle Name:

* Last Name:

Suffix:

3. Applicant Organization Contact

Person to be contacted on matters involving this application

* Phone Number: Fax Number:

Email:

OMB Number: 0925-0001 

Province:

Prefix:

Dr. Kenneth

Coleman

No Yes

No Yes

Dr. Michael

LaFleur

Chief Operating Officer

650 Albany ST

Boston

USA: UNITED STATES

MA: Massachusetts

02118-2518

	


 		

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Clinical Trial & HESC Page 58

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
4. Human Embryonic Stem Cells

* Does the proposed project involve human embryonic stem cells?

If the proposed project involves human embryonic stem cells, list below the registration number of the 

specific cell line(s) from the following list: http://stemcells.nih.gov/research/registry/. Or, if a specific 

stem cell line cannot be referenced at this time, please check the box indicating that one from the 

registry will be used: 

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

PHS 398 Cover Page Supplement

No Yes

Cell Line(s):

	


 		

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Clinical Trial & HESC Page 59

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
PHS 398 Research Plan

1. Application Type:

From SF 424 (R&R) Cover Page. The response provided on that page, regarding the type of application being submitted, is repeated for your 

reference, as you attach the appropriate sections of the Research Plan.

*Type of Application:

OMB Number: 0925-0001 

2. Specific Aims

3. *Research Strategy

6. Protection of Human Subjects

7. Inclusion of Women and Minorities

8. Targeted/Planned Enrollment Table

9. Inclusion of Children

10. Vertebrate Animals

13. Consortium/Contractual Arrangements

14. Letters of Support

15. Resource Sharing Plan(s)

16. Appendix

1. Introduction to Application 

(for RESUBMISSION or REVISION only)

Human Subjects Sections

2. Research Plan Attachments: 

 Please attach applicable sections of the research plan, below.

Other Research Plan Sections

5. Progress Report Publication List

11. Select Agent Research

12. Multiple PD/PI Leadership Plan

4. Inclusion Enrollment Report

New Resubmission Renewal Continuation Revision

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List of Research Plan Attachments Page 60

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
We are encouraged by the generally positive evaluation of our proposal: “Significance resides in addressing an 

area of unmet medical need for novel therapeutics that overcome issues with recalcitrant infections and are 

effective against dormant persisters and biofilm growth...” “The outstanding investigator is highly qualified to 

lead a very strong team that is highly experienced in drug development and discovery...” “Strengths of this 

application are the clever, innovative mechanism of action of ADEP4 to activate dormant cells to increase 

antibiotic sensitivity and the preliminary results that most reviewers regarded as strong and promising as they 

reveal combinations of ADEP4/antibiotic are effective against biofilms and stationary bacteria.” ” Overall, there 

is very strong confidence in the likelihood of this project’s success and that the proposed work will lead to 

development ADEP-antibiotic combinations as effective candidate therapeutics for recalcitrant S. aureus 

infections.” – Summary Statement. 

We are grateful to the Reviewers for their constructive comments that allowed us to prepare a stronger 

proposal. Our replies to critiques follow. Changes in the proposal have been italicized. 

Reviewer I. 1. The background on combination therapeutics for treating S. aureus infections is too narrative. –

We include specific data on combinations in the revision. 

2. Development of combination therapeutics for drug-resistant pathogens is not new. Using a ClpP activator, 

ADEP4 a derivative of ADEP is not new. – In the revised application, we note that existing combinations do not 

eradicate biofilm infections. Regarding ADEP4, several studies suggested that ADEP4/ClpP only attacks 

targets in growing cells. We show that ADEP4 activates proteolysis in non-growing cells, and detail this in the 

revision. 

3. It is unclear whether the combination treatment eradicated S. aureus from infected thighs or still had 3 log 

CFU (Fig. 4B). – There were no remaining CFUs suggesting sterilization and we clarify this in the revision. 

4. Lack of rationale to repeat to combine ADEP4 with rifampicin and linezolid in Aim 1. – Agreed, redundant 

repetition eliminated, section rewritten. 

5. Lack of detailed information of how many S. aureus isolates will be utilized for the validation studies. –

Information provided. 

6. Lack of detailed critical information regarding in vitro ADME/DMPK/toxicity studies in Aim 1. – Information 

provided. 

7. The Reviewer notes that “This phase I application has clear, appropriate, measurable goals (milestones) 

that should be achieved prior to initiating Phase II.”, but also “However, it is unclear when the applicant is able 

to obtain letters of interest, additional funding commitments, and/or resources from the private sector or nonSBIR/STTR funding sources that would enhance the likelihood for commercialization.”, and concludes that fast 

track is therefore unacceptable. Our plan is to enter into partnership once strong data from Phase II are 

available. Based on our considerable experience with Pharma and Biotech, it is unrealistic to obtain a 

meaningful commitment from a partner at this early stage of development. 

Reviewer II. 1. ADEP compounds were previously published by other companies many years ago. – Please 

see reply 2, Reviewer I. 

2. It is unclear if ADEP has the potential problems common to peptide antibiotics, such as solubility, in vivo 

stability and other undesirable pharmacokinetics. Another unclear is if the uncontrolled proteolysis due to an 

ADEP-activated ClpP causes any undesirable effect, in addition to the desirable activation of the dormant cells 

biofilms and dormant cells. – Preliminary pharmacokinetics show that the drug achieves a reasonable level, 

and this is confirmed by the complete sterilization of a biofilm infection within 24 hours. This is also in 

agreement with sterilizing results from a hollow fiber model. Rapid sterilization resulting in a very short duration 

of treatment is likely to diminish any potential side-effects. 

3. The project relies on outsourced synthesis of compounds and pharmacology, toxicology, and other tests. 

-The total amount of outsourced work is well below the 50% SBIR limit. It represents only ~13% of the budget. 

4. VAS does not address the use of rats use of mice is appropriately described. – VAS modified 

accordingly. 

5. Biohazards: Protections are not adequately described. – Additional biohazard and PPE details provided. 

6. Recommended reductions in 3 Research Associates (Year 2 and Year 3) and 1 Research Associate (Year 

4) as these are not well justified. - These reductions were made as recommended. 

Reviewer III. 

1. VAS on use of rats is not provided while use of mice is well-described. – Please see reply 4, 

Reviewer II. 

2. Recommended reductions in 3 Research Associates (Year 2 and Year 3) and 1 Research Associate (Year 

4) as these positions are not well justified. – Please see reply 6, Reviewer II.

Introduction Page 61

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
The goal of this project is to develop the antibacterial ADEP to treat recalcitrant S. aureus infections. This 

molecule is uniquely capable of sterilizing biofilm-related recalcitrant infections, such as abscesses, 

osteomyelitis and endocarditis. These infections are essentially untreatable and lead to substantial morbidity 

and mortality. In many cases, recalcitrance is not caused by drug resistance, but by the inability of current 

antibiotics to target the cells within a biofilm. Indeed, biofilms contain heterogeneous populations of stationary, 

slowly growing, non-growing, and dormant persister cells, which are highly tolerant to killing by traditional 

antibiotics (Lewis, 2010). When the antibiotic concentration drops, these cells are able to regrow and 

repopulate the biofilm. In S. aureus, a stationary population is completely refractory to killing by traditional 

antibiotics. Bactericidal antibiotics require an active target to be corrupted in order to kill. For example, the 

fluoroquinolones turn DNA gyrase into an endonuclease and the aminoglycosides cause mistranslation. This 

approach does not work on cells within a biofilm, which contain a high proportion of non-growing cells. Biofilms 

are protected from the immune system, compounding the problem. We reasoned that dormant cells could be 

targeted by an antibiotic which causes killing through the activation of a target. Acyldepsipeptide (ADEP) was a 

possible candidate for such a compound. ADEP causes dysregulation of the ClpP protease, relieving it from 

regulatory control and the requirement to use ATP. However, ADEP/ClpP was reported to only attack targets in 

growing cells. We reexamined this and find that ADEP induces massive proteolysis in stationary cells of S. 

aureus, forcing them to self-digest (Conlon et al., 2013). We showed that ADEP has a remarkable ability to kill 

persisters and stationary cells. 

ADEP has been previously examined and was effective in mouse models of uncomplicated bacteremia, 

however conventional antibiotics are highly effective in these models as well. ADEP has a liability – ClpP is not 

essential, and a high-frequency of null mutations leads to resistance. For this reason, development of ADEP4 

was terminated. However, knowing the potential of ADEP to target dormant cells, it makes sense to combine it 

with conventional antibiotics, which will solve the resistance problem. Indeed, combining ADEP4 with rifampicin 

or linezolid produced complete sterilization of stationary and biofilm S. aureus cultures. Importantly, ADEP4 in 

combination with rifampicin sterilized MRSA in a mouse model of deep-seated infection, where conventional 

antibiotics had very little effect. In Phase I of this project, we will identify the best ADEP partnering antibiotic 

and we will evaluate the combinations for the potential to develop resistance in the hollow-fiber model. Phase II 

will focus on in vivo PK, efficacy and pre-IND enabling studies. 

This project is a result of a collaboration between the PI, Dr. Ken Coleman, an expert in drug discovery 

and development (Arietis), Kim Lewis, an expert in antimicrobial resistance and tolerance (NU) and Steven 

Leonard, an expert in the hollow fiber model and pharmacology (NU).

Phase 1 Segment- Aim 1. Identify the best sterilizing combination of ADEP4 and an approved 

antibiotic. ADEP4 will be paired with known antibiotics such as daptomycin, gentamicin, and oxacillin, and 

examined for its ability to sterilize stationary and biofilm populations of S. aureus. ADEP/antibiotic 

combinations will also be tested for maximum killing and low resistance development using a hollow-fiber 

model of in vitro infection. Milestone: Advance 2 ADEP4/antibiotic combinations to in vivo studies. 

Combinations which effectively sterilize biofilms and stationary populations of S. aureus, and have a rate of 

resistance development <10-9 in the hollow fiber model will be advanced. 

Phase 2 Segment- Aim 2. In vivo testing of sterilizing combinations. PK parameters and initial efficacy of 

two ADEP4/antibiotic combinations will be determined in mice. The PK/PD driver of ADEP efficacy will be 

determined using the mouse model of deep-seated MRSA infection. Efficacy of each combination will be tested 

in an implanted tissue cage biofilm model. Milestone: Determine the PK of the combined agents, the 

pharmacodynamic driver of efficacy, and treat a previously incurable biofilm MRSA infection. 

Aim 3. Preclinical development. Acute and dose-range finding toxicity studies will be performed in rats 

. A 14 day repeated dose toxicity study will be performed in rats. PK studies in rats will provide 

the basis for selecting doses to be tested in humans. Ames and chromosomal aberration genotoxicity studies 

will also be performed. Taken together, these studies will form the basis for a risk-benefit assessment prior to 

the pre-IND meeting with the FDA and entry into clinical trials.

Specific Aims Page 62

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Research Strategy 

(a) Significance. 

The goal of this project is to develop a therapeutic capable of treating chronic drug-tolerant infections. While 

drug resistance is a formidable challenge, there is a considerable arsenal of antibiotics that are effective 

against most disseminated infections, and new compounds are slowly moving through the drug discovery 

pipelines. The situation with tolerance is very different – there has never been a therapeutic capable of 

sterilizing a chronic infection. Antibiotics are effective because of cooperation with the immune system. An 

antimicrobial kills or slows the growth of the pathogen, and the immune system cleans up the rest. But this 

does not work when an exopolymer matrix restricts access of the immune system to the pathogen within a 

biofilm. The result is a chronic infection, requiring treatment with multiple antibiotics over the course of months 

to years, accompanied by significant morbidity and mortality. These include abscesses, deep-seated 

infections, endocarditis, osteomyelitis, cystic fibrosis, infections of indwelling devices, and dental diseases. 

Only recently did we gain insight into the cause for the limited efficacy of antibiotics against chronic infections. 

All communities of cells produce persisters, dormant variants of the wild type which are tolerant to antibiotics 

(Lewis, 2010). In order to kill, an antibiotic requires an active target. Aminoglycosides cause mistranslation, 

which creates toxic misfolded proteins; fluoroquinolones convert DNA gyrase/topoisomerase into an 

endonuclease, which cleaves DNA; and β-lactams activate autolysins that destroy the cell wall. Drug targets 

are largely inactive when a cell is dormant, and antibiotics fail (Keren et al., 2004b). Once the concentration of 

an antibiotic drops, persisters repopulate the biofilm, causing a relapsing chronic infection.

What is known about persisters can be summarized as follows: persisters are not resistant mutants but 

phenotypic variants of regular cells produced stochastically in the population (Keren et al., 2004a); all 

pathogens form persisters (Lewis, 2010); the relative abundance of E. coli persisters is higher in lateexponential and biofilms, reaching 1% (Keren et al., 2004a); toxin-antitoxin modules (TA) contribute to 

persister formation (Keren et al., 2004b, Schumacher et al., 2009, Dorr et al., 2010); persisters are nongrowing (Balaban et al., 2004), dormant (Shah et al., 2006) cells, which explains their tolerance to bactericidal 

antibiotics and their role in recalcitrance of disease (LaFleur et al., 2010, Mulcahy et al., 2010); pathways of 

persister formation are highly redundant (Hansen et al., 2008, Maisonneuve et al., 2011). This presents an 

obvious problem – persisters cannot be eliminated with traditional antibiotics, or by the immune system if the 

cells are located within the biofilm. 

It was recently suggested that adding sugars such as glucose or mannitol to a stationary culture will 

resuscitate persisters which can then be killed by aminoglycosides (Allison et al., 2011). Given that the 

bloodstream has an abundant supply of glucose, the practical implications of this result are unclear. A recent 

report described a compound resulting from an HTS that potentiated norfloxacin, completely killing an 

exponentially-growing culture of E. coli (Kim et al., 2011). This is an interesting observation, but conventional 

antibiotics do slowly kill E. coli persisters formed in an exponential culture as well. The challenge is to sterilize 

a stationary or biofilm culture. Influx tried finding potentiators of bactericidal antibiotics that kill stationary cells 

by screening a synthetic compound library but failed (Penny Markham, personal communication). There was 

also a claim that all bactericidal antibiotics kill by forming reactive oxygen species, and activating ROS 

formation or suppressing protective mechanisms will eliminate tolerant cells (Kohanski et al., 2007, Dwyer et 

al., 2009, Nguyen et al., 2011). These finding were recently refuted (Liu & Imlay, 2013) and our careful side-byside examination showed virtually no difference in MBC under aerobic vs. anaerobic growth (Keren et al., 

2013). The problem of drug-tolerant cells is highly significant to human health, and the challenge of eliminating 

persisters is formidable. 

(b) Innovation. 

We reasoned that activation of a target, rather than inhibition, could lead to the killing of dormant cells. Indeed, 

there is an antimicrobial compound, acyldpepsipeptide (ADEP), which activates the ClpP protease, relieving it 

both from regulatory control and from the requirement to use ATP (Brotz-Oesterhelt et al., 2005). The ClpP 

protease is fairly selective, clearing misfolded proteins that are fed into the enzyme by accessory ClpA, ClpC 

and ClpX subunits, which use ATP to recognize, unfold and translocate substrates into the ClpP proteolytic 

chamber for degradation. ADEP binds to the entry pocket of ClpP, keeping the proteolytic chamber open (Lee

et al., 2010). The result is a non-specific protease that does not require ATP. However, ADEP/ClpP was 

reported to only attack targets in growing cells. Nascent polypeptides emerging from the ribosome, rather than 

mature folded proteins, were reported to be targets of ADEP4/ClpP (Kirstein et al., 2009). This would indicate 

that ADEP4 targets growing cells with active protein synthesis. A particular mature protein, FtsZ, has been 

reported to be a major target of ADEP4/ClpP (Sass et al., 2011). FtsZ forms the cell division ring, suggesting 

Research Strategy Page 63

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
activity of ADEP4 against growing cells as well. We noted that the studies were performed with very short time 

exposures, and decided to re-examine the action of ADEP. We found that ADEP induces massive proteolysis 

in stationary cells of S. aureus, forcing them to self-digest (Conlon et al., 2013). Importantly, ADEP was able to 

effectively sterilize a culture of stationary phase S. aureus (Preliminary Studies). The only other antibiotic that 

has activity against stationary S. aureus is the membrane-acting agent daptomycin. At high concentrations 

daptomycin is active against stationary S. aureus in vitro (Murillo et al., 2009), but it does not sterilize at 

clinically achievable concentrations. Daptomycin is the best available bactericidal compound, but treatment 

failures do occur and they may be caused by its limited activity against persisters at therapeutic concentrations 

(Lamp et al., 2007). The exceptional killing ability of ADEP opens the possibility to develop the first therapeutic 

which specifically targets recalcitrant chronic infections. 

Acyldepsipeptide is produced by a soil streptomycetes and was discovered 26 years ago by scientists 

from Eli Lilly (Michel & Kastner, 1985). They evaluated the compound, found that it had activity against grampositive but not gram-negative bacteria, and dropped it. Twenty years later, there was a serious need to treat 

infections caused by drug resistant gram-positive pathogens. By that time Eli Lilly was no longer in the 

business of antiinfectives, and Bayer picked up acyldepsipeptide for evaluation. They produced a more active 

derivative, ADEP4, and determined its mechanism of action (Brotz-Oesterhelt et al., 2005). Importantly, 

ADEP4 was found to be safe and effective in animal models of uncomplicated infection caused by S. aureus, 

S. pneumoniae and E. faecalis. However, resistance development to ADEP4 was high due to null mutations in 

ClpP, and the compound was dropped again. Bayer has since closed its antiinfective research division and its 

patent for ADEP4 has lapsed. Knowing that ADEP has the potential to sterilize infections, we will combine it

with conventional antibiotics to solve the resistance problem. 

Combining ADEP with antibiotics such as rifampicin or linezolid produced complete sterilization of 

stationary S. aureus cultures. Given that a stationary population contained 109 cells, and clpP mutants arise 

with a frequency of 10-6, we expected to see 103 surviving cells in this experiment. Unexpectedly, we found that 

ClpP mutants become highly susceptible to killing by most antibiotics, explaining the sterilizing effect of the 

combination seen in vitro. ADEP + rifampicin also completely sterilized a deep-seated biofilm infection of 

MRSA in neutropenic mice (Preliminary Studies). Vancomycin or rifampicin had very little effect in this model. 

(c) Approach. 

Preliminary Studies. Previous studies showing that ADEP targets nascent peptides and FtsZ in particular 

were performed with short exposure times and with rapidly growing cells, and we considered the possibility that 

longer incubation with ADEP may result in nonspecific degradation of proteins in non-growing cells. A 

stationary phase population of S. aureus was chosen to test this, as cells are not dividing and synthesis of 

nascent polypeptides is strongly down regulated (Michalik et al., 2009). Stationary cells of methicillin-resistant 

S. aureus (MRSA) were exposed to ADEP4 for 24 h and the resulting proteome was compared with that of an 

untreated control (Fig. 1). 

 Proteomic analysis of untreated stationary cells led to the detection of 1,712 proteins representing 65% 

of the predicted open reading frames. Treatment with ADEP4 resulted in decreased abundance of 243 proteins 

(P ≤ 0.05 and twofold decrease) (Fig. 1A). Essential ribosomal proteins were among the most strongly 

diminished by ADEP4/ClpP, with proteins S21, L9, S1 and ribosomal recycling factor all showing between 17- 

and 64-fold reduction in the ADEP4 treated sample. Elongation factor Tu, pyruvate kinase and fructose biphosphate aldolase were among the proteins with the largest increase in non-trypsin cleavage sites. FtsZ was 

also one of the many strongly degraded proteins perhaps because of its disordered carboxy terminus (Buske & 

Levin, 2013). Other than the ribosome, degraded proteins belonged to various functional types, including 

purine metabolism, glycolysis and aminoacyl-tRNA biosynthesis, among others (Fig. 1B). Taken together, the 

proteomic data indicated that ADEP4 forced the cell to self-digest, suggesting that it could be effective in killing 

dormant cells. 

Research Strategy Page 64

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
The natural product acyldepsipeptides are not particularly potent, but a medicinal chemistry program 

conducted by Bayer resulted in ADEP4, with an IC50 of 0.05 μg/ml against S. aureus. The killing ability of 

ADEP4 had not been examined. Another analog, ADEP-10c was subsequently described by a different group, 

with a reported MIC of 0.6 μg/ml (Socha et al., 2010). We decided to test the killing properties of both 

compounds. In our hands, ADEP 10c had a S. aureus MIC of 5 μg/ml, and ADEP4 had an MIC of 0.75 μg/ml. 

Since the Bayer group only reported IC50 rather than MIC, our results are probably in accordance with their 

findings. ADEP 10c is considerably less active; apart from the compounds synthesized for this project, we also 

tested a sample from the authors (Socha et al., 2010), and got the same MIC, 5 μg/ml. We therefore decided 

to focus on ADEP4. ADEP4 had a MBC against S. aureus of 0.75 μg/ml, which is identical to the MIC and a 

good indication of its excellent killing ability. 

As mentioned previously, ADEP4 uncouples ClpP from the requirement to use ATP, which would help 

kill persisters with low energy levels. In a control experiment, ciprofloxacin was added to an exponentially 

growing culture of S. aureus, which produced a typical biphasic killing pattern with surviving persisters (Fig. 

2A). 

2

3

4

5

6

7

8

9

10

0 1 2 3 Log (cfu/ml)

Time [days]

Control

ADEP 4

Rifampicin

ADEP4

Linezolid

ADEP4

Ciprofloxacin

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 Log (cfu/ml)

Time [days]

Control

Rifampicin

Linezolid

Vancomycin

Ciprofloxacin

1

2

3

4

5

6

7

8

0 2 4 6 24 48

Log (cfu/ml)

Time [h]

Ciprofloxacin

Ciprofloxacin then

Rifampicin

Ciprofloxacin then

ADEP4

A B C

D

2

3

4

5

6

7

8

9

10

0 1 2 3 Log (cfu/ml)

Time [days]

ADEP4

Control

2

nd Antibiotic 

added

E

Figure 2. ADEP4 kills persisters. A) ADEP4 

kills persisters surviving ciprofloxacin 

treatment. B) Conventional antibiotics are 

inactive against stationary phase S. aureus. 

C) Combinations of conventional antibiotics 

against stationary phase S. aureus. D) 

ADEP4 activity against stationary S. aureus. 

E) ADEP4 in combination with rifampicin, 

linezolid or ciprofloxacin eradicates 

stationary phase S. aureus to the detection 

limit in 72 h in MHB.

A B

Figure 1. Proteomic analysis of S. aureus cells treated with ADEP4 reveals extensive protein degradation. A) The dispersion 

graphs show the relative abundances (treated/untreated) of total proteins in different biological replicates (n = 2). The significant 

changes in abundances (P ≤ 0.05 and >twofold) are represented in red circles. B) Function–enrichment analysis of proteins 

degraded by ADEP4. Functions overrepresented among proteins degraded by ADEP4 were annotated using Database for 

Annotation, Visualization and Integrated Discovery (DAVID) and the overrepresented pathways compared to the genome 

background are shown as columns, whereas their P-values are represented by the black dots. Bayesian moderated t-test was 

used to provide P-values that were further corrected by the data set size.

Research Strategy Page 65

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Addition of rifampicin to surviving persisters had no effect on their viability, in agreement with previous 

observations on the multidrug tolerant nature of these cells. By contrast, addition of ADEP4 led to eradication 

of persisters (Fig. 2A). In agreement with our previous observations (Keren et al., 2004a), high concentrations 

(10x MIC) of bactericidal antibiotics vancomycin, ciprofloxacin, rifampicin or linezolid had little effect on a 

stationary population of S. aureus cells even after a 5 day incubation (Fig. 2B). Furthermore, combinations of 

vancomycin, rifampicin and ciprofloxacin had very limited activity (Fig. 2A, 2C, 6B, and 7B). ADEP4 at 1x MIC 

produced much more killing after a single day of incubation, decreasing cell numbers of a stationary population 

by 4 logs (Fig. 2D). After that, the culture partially rebounded, due to the appearance and growth of clpP

mutants, which were validated by PCR and sequencing. However, combining ADEP with rifampicin, linezolid or 

ciprofloxacin produced complete sterilization by day 3 (Fig. 2E). 

Complete sterilization in these experiments was unexpected—the frequency of clpP mutants is 10−6, 

and in a population of 109 cells, there should have been 

103 survivors. To investigate this, a ΔclpP mutant was 

examined for its susceptibility to linezolid, rifampicin and 

ciprofloxacin (Fig. 3). The ΔclpP strain had the same 

minimum inhibitory concentration (MIC) as the wild type, 

but stationary phase counts were reduced 10- to 100-fold 

upon exposure to linezolid, rifampicin or ciprofloxacin. A 

mutation in clpP apparently diminishes the fitness of cells 

and makes them vulnerable to certain antibiotics. In 

agreement with this, a clpP mutant was reported to be 

avirulent in a murine skin abscess model of infection 

(Frees et al., 2003). 

We then tested the eradicating potential of the 

ADEP4 and rifampicin combination against a variety of S. 

aureus strains. These included the laboratory strain 

SA113, as well as clinical isolates USA300, UAMS-1 and strain 37. USA300 is a community acquired MRSA 

and is the most common cause of staphylococcal skin and 

soft tissue infections in the United States(Stryjewski & 

Chambers, 2008). UAMS-1 is a highly virulent clinical 

isolate associated with chronic osteomyelitis (Gillaspy et 

al., 1995). Strain 37 was isolated from a patient undergoing 

vancomycin therapy who succumbed to infection (Miyazaki 

et al., 2011). No colonies were detected in any of these 

strains after 72 h of incubating stationary cultures with the 

combination of ADEP4 and rifampicin (Fig. 4). 

 Based on these encouraging results, we decided to 

test the antibiotics against biofilms. Biofilms of S. aureus

UAMS-1 were grown overnight in 96-well plates in BHI 

broth. Spent media was replaced with fresh BHI containing 

10x MIC of various antibiotics and incubated for 1 or 3 

days. Wells were washed with PBS and the biofilms were 

disrupted with sonication and plated on BHI agar for colony 

counts. As expected, conventional antibiotics had little 

effect on the biofilm (Fig. 5A). A combination of ADEP4 

with rifampicin resulted in eradication of cells in the biofilm 

to the limit of detection (Fig. 5A). The replacement of 

antibiotics with fresh medium did not result in re-growth 

after 3 days of ADEP/rifampicin treatment, confirming the complete eradication of living cells. Confocal 

microscopy also showed that the combination of ADEP + rifampicin eradicated the biofilm (Fig. 5B). An 

elimination of a biofilm is unprecedented for such low, clinically achievable concentrations of compounds. 

Figure 3. clpP mutants are susceptible to killing by 

antibiotics. Stationary cultures of S. aureus were 

exposed to 10x the MIC of each antibiotic for 2 days. 

Figure 4. S. aureus was grown in MHB for 16 h 

and challenged with 10× MIC of ADEP4 and 

rifampicin. Colony counts were performed every 

24 h. The x axis is the limit of detection. Data are 

representative of 3 independent experiments. 

Error bars represent standard deviation.

Research Strategy Page 66

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
 Eradication of stationary and biofilm populations was an encouraging sign that ADEP4 could be a very 

useful antibiotic against untreatable chronic infections. To test this, we used a deep-seated mouse thigh 

infection model. In a standard thigh model, a mouse is infected with a low dose of pathogen and antibiotic 

therapy begins within a few hours of infection. Under these conditions, conventional antibiotics are very 

effective. In the deep-seated model, the mouse is made neutropenic by treatment with cyclophosphamide, a 

large dose of pathogen is delivered and the infection is allowed to develop for 24 h before therapy, leading to a 

severe, recalcitrant, deep-seated infection. This model emulates a difficult to treat human deep-seated chronic 

infection in immunocompromised patients. We performed histopathology of the infected thigh, and detected 

massive aggregates of S. aureus cells with Gram staining (Fig. 6A). 

A B

control

ADEP + 

rifampcin 

Figure 5. The effect of antibiotics on the survival of S. aureus biofilms. A) Biofilms were grown in 96 well plates and 

exposed to antibiotics at concentrations of 10x the MIC for 24 and 72 hours. B) Confocal microscopy live cell imaging of 

a control biofilms or one treated with the combination of ADEP and rifampicin at 1 and 10 x the MIC for 72 hours. S. 

aureus strain UAMS-1 was transformed with a GFP plasmid under the control of xylose inducible promoter. Biofilms 

were seeded and grown in an imaging flow cell for 24 hours. After drug exposure, the biofilms were washed with fresh 

medium containing xylose to induce GFP expression in live cells.

Figure 6. In vivo model of deep seated S. aureus infection. A) Gram staining and electron microscopy histopathology 

cross-sections of infected thigh tissue harvested 24h post infection. Tissues were fixed in 10% formalin or EM fixative 

(paraformaldehyde/glutathione) prior to processing and imaging. B) In vivo activity of antibiotics in a deep seated 

neutropenic mouse thigh lesion model. Groups of 5 mice were infected with 1x 106 cells of S. aureus MRSA ATCC 33591 

and infection was allowed to develop for 24 hours. Vancomycin was administered at 110 mg/kg twice a day. Rifampicin 

and ADEP were administered once at 30 mg/kg and 50 mg/kg respectively alone or in combination. Mice were 

euthanized and the thigh muscle was removed aseptically and homogenized in PBS. Serial dilutions were spotted on 

MHA and incubated overnight at 37oC. *No cell counts were present in the ADEP + rifampicin group, even when the 

entire thigh homogenate from each animal was plated.

A

40X

160X

320X

Gram 

stain

biofilm 

abscesses

individual 

cells

EM 

4000X

biofilm 

matrix

B

*

Research Strategy Page 67

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Electron microscopy of cross-sections of the infected tissue revealed S. aureus growing in biofilms adhered to 

muscle cells (Fig. 6A). Administration of vancomycin, rifampicin or a combination of both decreased the viable 

counts, but did not clear the infection (Fig. 6B). Furthermore, no notable difference was observed between 

mice treated for 24 h or 48 h with vancomycin in this model, indicating the presence of a persister 

subpopulation surviving the antibiotic treatment (Conlon et al., 2013). Remarkably, an ADEP4 and rifampicin 

combination led to sterilization of the infected tissue within 24 h (Fig. 6B). No colonies were present even when 

the entire thigh homogenate from each animal was plated. Based on this efficacious dose and our mouse 

pharmacokinetics data (Fig. 7A), we performed a hollow-fiber experiment and found that the combination of 

ADEP and rifampicin also resulted in complete eradication of the pathogen to the limit of detection in these 

experiments (Fig. 7B). Complete sterilization of a chronic infection with a single dose of an antimicrobial 

combination bodes well for development of an effective therapeutic to target difficult to treat chronic infections. 

Aim 1. Identify approved antibiotics to combine with ADEP4 and validate the combination treatment. 

So far, we have tested a combination of ADEP4 with rifampicin; linezolid; and ciprofloxacin. For complete 

characterization, we will also examine the remaining main classes of antibiotics, a β-lactam (oxacillin), an 

aminoglycoside (gentamicin), and a lipopeptide (daptomycin). Again we will test osteomyelitis isolate UAMS-1, 

MRSA strains ATCC33591 and community acquired USA300, and a clinical isolate which was responsible for 

death of a patient undergoing vancomycin therapy (Miyazaki et al., 2011). Combinations will be tested at 

clinically achievable concentrations against stationary cultures of these pathogens. Complete sterilization will 

indicate a good combination. We will then examine combinations that pass this test against biofilms. For 

testing biofilms, we will use two different models. One is a CalgaryTM device with prongs which is placed in a 

suspension of bacteria in nutrient medium for the biofilms to form. Another method is based on forming a 

biofilm in a flow cell, which probably better resembles an environment in vivo due to shear flow. The 

osteomyelitis isolate UAMS-1 will be used for biofilm testing. As a final validation, ADEP/antibiotic 

combinations will be tested for maximum killing and low resistance development using an in vitro hollow-fiber 

infection model. The MRSA strains ATCC33591 and community acquired USA300 strains will be used for the 

hollow-fiber model. Since we are registered with the Network on Antimicrobial Resistance in Staphylococcus 

Aureus (NARSA), and have access to their strain collection, newly emergent isolates which are deposited in 

their repository will also be available for testing. 

The reason for using combinations is to minimize the probability of resistance. As mentioned, null clpP

mutants resistant to ADEP4 arise with a probability of 10-6. As we report, these mutants are highly attenuated; 

they lose virulence and become susceptible to killing by other antibiotics. The frequency of rifampicin 

resistance is around 10-8, which suggests that mutants resistant to the combination will arise with a frequency 

of 10-14. Resistance development for other antibiotics is <10-9, suggesting that the combination will have an 

even greater safety margin. So far, we have not seen spontaneous resistant mutants develop for the 

combination of ADEP/rifampicin tested at 10X the MIC on agar plates. However, resistance may occur as a 

result of changing drug concentrations and exposures. Therefore we will determine the relationship between 

A

1

10

100

1000

10000

0 2 4 6 8

Concentration (ng/mL)

Time (h)

B

Figure 7. A) ADEP concentrations in the hollow fiber experiment were based on the AUC calculated from free drug 

plasma concentrations in mice. B) Survival of S. aureus exposed to antibiotics for 4 days in a hollow-fiber infection 

model. Antibiotic concentrations were based on physiologically relevant concentrations achieved in humans and were 

adjusted for protein binding. No cell counts were present after 24 hours.

A

Research Strategy Page 68

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
ADEP4/antibiotic exposure and drug resistance in the hollow-fiber infection model. ADEP4 will be paired with 

known antibiotics such as daptomycin, linezolid, ciprofloxacin, gentamicin, and oxacillin, and examined for the 

ability to sterilize and prevent resistant population take-over in the hollow fiber infection model. The hollow-fiber 

model is a pharmacodynamic system that allows a pathogen to grow in a chamber within a hollow-fiber 

cartridge. The bacterial growth chamber is separated from the central compartment by a semi-permeable 

membrane, which allows nutrients and drugs to pass through, while retaining the cells. Fresh media and drugs 

are pumped into the central compartment allowing different half-lives and exposure conditions to be simulated, 

without diluting the pathogen. Sample ports allow drug concentrations and culture densities to be monitored. 

We have performed a preliminary experiment using this model in order to validate this approach (Fig. 

7B). Antibiotic concentrations will be calculated and delivered based on known standard dose regimens in 

humans, adjusted for each antibiotic half-live and the extent of protein binding. ADEP concentrations were 

calculated in a similar fashion based on a preliminary determination of PK in mice. The ADEP drug 

concentration vs time area under the curve (AUC) was determined based on a 5 mg/kg dose delivered by IV 

tail vein injection (Fig. 7A). The AUC was multiplied by 10 to estimate the 50 mg/kg dose which was effective in 

vivo (Fig. 6B), assuming a linear correlation, and adjusted for plasma protein binding. These experiments 

mimicked the in vivo results with the neutropenic murine thigh model of deep seated MRSA infection. The 

combination of ADEP and rifampicin caused complete sterilization after 24 hours and no resistance occurred 

over 4 days (Fig. 7B). Resistance to rifampicin occurred within 24 hours, while dosing with vancomycin or 

vacomycin + rifampicin achieved a steady state, 2 log reduction in colony counts. 

In Aim 1, a more detailed analysis of resistance will be performed. The experiments will be expanded to 

10 days in duration and the frequency of resistance development for 3 different ADEP4/partner antibiotic ratios 

will be determined. The relationship between drug exposure and resistance is an inverted-U. Resistance is less 

for low and high drug exposures compared to intermediate levels. Therefore, several doses of ADEP will be 

evaluated. The published maximum tolerated dose of ADEP4 is 100 mg/kg (Brotz-Oesterhelt et al., 2005) and 

50 mg/kg sterilized in the mouse model. Rifampicin was dosed at 30 mg/kg once per day for the S. aureus 

infection model, which mimics the 600 mg daily dose in humans. Therefore, ADEP will be added at 1:1, 2:1 

and 3:1 dosing ratios (30, 60 and 90 mg/kg). Samples will be taken from the bacterial chamber, washed, 

diluted, and plated to determine the total live bacterial population for each time point. A portion of each sample 

will also be plated on agar containing 3 x MIC of each drug, in order to determine whether a resistant 

subpopulation exists and at what level. Similar experiments will be repeated for each antibiotic - linezolid, 

ciprofloxacin, gentamicin, daptomycin and oxacillin. Dosing for each known antibiotic will mimic the standard 

human dose and three different concentrations of ADEP4 will be tested for each antibiotic. Combinations with a 

resistance rate less than 10-9 will be considered acceptable for further advancement. Ultimately the 

ADEP4/antibiotic combinations with the best killing potential and lowest frequency of resistance will emerge. 

In vitro ADME/DMPK/Toxicity. The potential for secondary drug interactions will be examined by testing 

ADEP4 in combination with each partner antibiotic against a safety panel of human enzymes and receptors 

using the Eurofins lead profiling + CYP screening. This screen detects potential adverse activity against 68 

primary molecular targets and the five major CYP450s. The full list of the enzymes to be tested is available 

from Eurofins and includes human adenosine, adrenergic, bradykinin, calcium channel, dopamine, GABA, 

glutamate, histamine, muscarinic, opiate, potassium channel, purinergic and serotonin receptors. 

 This data supports other published findings regarding the excellent safety of ADEP4 (BrotzOesterhelt et al., 2005). A combination of ADEP4 and each antibiotic will be tested in duplicate at 10 μM and 

screened for inhibition >50% compared to negative controls. Any hit will be confirmed in a dose response 

follow-up experiments comparing the activity of each component to the combination. Any combinations 

showing significantly higher adverse activitiy compared to each agent alone will be deprioritized. 

Recent reports have shown that ADEPs are able to activate human mitochondrial hClpP in vitro (Lowth 

et al., 2012). However, toxicity associated with unregulated protein degradation in mitochondria has not been 

observed either in cultured eukaryotic cells or in animal studies (Brotz-Oesterhelt et al., 2005). Preliminary 

pharmacokinetics and efficacy in mice shows that ADEP achieves a reasonable level, confirmed by the 

complete sterilization of a biofilm infection within 24 hours. This is also in agreement with sterilizing results 

from a hollow fiber model. Rapid sterilization resulting in a very short duration of treatment is likely to diminish 

any potential side-effects. If we observe that a given combination of ADEP4 + antibiotic shows toxicity, this 

combination will be deprioritized. 

Research Strategy Page 69

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Pitfalls and Alternatives: We do not anticipate any problems with these studies as we have already 

validated these models. Additional approved antibiotics and higher or more frequent dosing regimens may be 

explored. 

Criteria for Phase II advancement and Fast-track justification. ADEP4 has a unique mechanism of action 

and an exceptional ability to kill stationary populations and biofilms. These properties suggest there is an 

untapped potential for ADEP based therapeutics to cure recalcitrant infection. In phase I, we will determine the 

best sterilizing combinations of ADEP/antibiotic. ClpP mutants are avirulent and highly susceptible to killing by 

traditional antibiotics, suggesting it is unlikely for resistance to the ADEP/antibiotic combination to occur. 

However, a more detailed study of resistance is warranted. We will therefore identify combinations that result in 

resistance frequency below 10-9. Milestone: Identify ADEP4/antibiotic combinations which sterilize MRSA 

in stationary, biofilm and hollow fiber models, and have a frequency of resistance development <10-9. 

ADEP4 is already known to be safe and effective in multiple animal models of uncomplicated infections. Our 

preliminary data show that ADEP and rifampicin cure a deep seated biofilm infection, while other antibiotics are 

ineffective. These findings suggest a realistic opportunity to develop an ADEP/antibiotic combination and justify 

our fast-track application. For purposes of clarity, a simplified project overview is given below. 

Phase 2- Aim 2. In vivo evaluation of sterilizing combinations. Evaluating multiple combinations will 

improve the chance for success. We expect most of the combinations to work well in vitro, and since the initial 

in vivo efficacy model is straightforward, 2 of the best combinations will be advanced. The goal of this aim is to 

determine, validate, and describe the parameters responsible for the in vivo efficacy of the combination 

therapeutic. In order to optimize dosing, it is necessary to determine the amount of each component to 

administer, and the frequency of administration. A determination of the pharmacokinetics (PK) and 

pharmacodynamics (PD) of the two agents is essential to enable this optimization. PK describes the changes 

in the plasma concentration of the drug over time, while PD describes the effect of the drug on the infecting 

organism. Although PD data can be collected in vitro, this may correlate poorly with in vivo PD data. For 

virtually all antiinfectives studied, the best predictor of efficacy, referred to as the PKPD driver, is one of the 

following parameters: 1) The area under the plasma concentration profile of unbound (free) drug (fAUC) 

compared to MIC (fAUC/MIC); 2) The duration within a 24h period for which the unbound (free) drug 

concentration exceeds the MIC (fT>MIC); or 3) The peak plasma concentration of unbound drug compared to 

MIC (fCMax/MIC). For example, if the PKPD driver of an agent is found to be a fAUC/MIC ratio of 100, and the 

infecting organism has an MIC of 4 μg/mL, the minimum efficacious dose is one which delivers a total 24h 

fAUC of 400 μg.h/mL. 

Figure 8. Overview and timeframe for the entire project. Phase 1 will be completed in 1 year, when the feasibility of 

developing ADEP4/antibiotic combination will be evaluated. Phase 2 will continue for 3 subsequent years and these studies 

will allow us to focus on a single antibiotic partner and enable a pre-IND meeting with the FDA.

Research Strategy Page 70

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
 Table 1. Mouse PK study design and animal numbers.

The PKPD driver for ADEP4 will be determined in combination with a fixed concentration of antibiotic 

using the methods described below. Since our primary aim for these combination agents is to eradicate an 

infection, these PKPD studies are critical for determining optimal dosing and efficacy. For example, if the 

PKPD driver proves to be fT>MIC, then drug dosing is optimized by prolonging the duration of exposure of the 

organism to the drug, either by administering more frequently at lower doses or by continuous infusion. If 

fAUC/MIC is the PKPD driver, then antimicrobial efficacy is optimized by the infrequent administration of large 

doses. The PKPD of the approved antibiotics are well defined and, by determining the PKPD driver for ADEP4 

in the presence of the antibiotic, it should be possible to optimize the ratio of the two drugs in combination, and 

determine the total daily dose of the combination and the dosing frequency. We will start by determining the PK 

of ADEP4 in combination with each antibiotic, and will then determine the PKPD driver of efficacy in the thigh 

model of deep seated MRSA infection. 

Pharmacokinetics. The PK of ADEP4 with antibiotic 1 or antibiotic 2 will be determined in mice (the PK of 

single agents is already known). PK will be determined for ADEP4 following IV and IP doing at 25, 50 and 100

mg/kg, all in combination with a 

standard dose of antibiotic 1 and 

antibiotic 2, using LC-MS/MS 

(Table 1). In the unlikely event of 

non-linear PK being detected, 

intermediate doses will also be 

studied. The oral bioavailability, 

elimination half-life, peak plasma 

concentration (CMax), area under 

the curve (AUC) and time above 

MIC (T>MIC) will be determined 

for each drug in the combination. 

These experiments will follow a 

typical 8-time point, single dose set-up. Serial micro- sampling from submandibular bleeds will be used to 

minimize mice and plasma will be collected at predetermined intervals of 15, 30, 45, 60, 90, 270, 480 and 1440 

minutes. The PK data will be used to adjust the dosing strategy for the initial efficacy studies, if necessary. 

Initial efficacy. We have previously used the neutropenic murine deep-seated S. aureus thigh infection as a 

model of recalcitrant infection. The murine staphylococcal thigh lesion model has been a standard in vivo test 

for antimicrobials for fifty or more years. In this model, an appropriate inoculum is introduced into the thigh 

muscle of the mouse to form an abscess. The infection remains localized 

for 4 or more days, causing enlargement of the thigh but no systemic 

infection. In most reported studies, chemotherapy commences within 2h 

of infection, as delaying therapy beyond this point usually results in failure 

to cure. In our hands, using immunocompromised mice and delaying 

therapy until 24h post-infection results in a highly recalcitrant biofilm 

infection, where even high doses of vancomycin or rifampicin dosed 

twice/day for 2 days gave less than a 2-log reduction in viable count 

relative to an untreated control. A single dose combination of rifampicin 

with ADEP4 cleared the infection. We will continue to use this recalcitrant 

abscess model to evaluate 2 ADEP4/antibiotic combinations. Initial 

studies will deliver 50 mg/kg of ADEP4 (1⁄2 of MTD) and the standard dose 

of the partner antibiotic, each adjusted based on the combined PK. Later 

studies will focus on optimizing the ratio of ADEP and finding the best 

dosing regimen for cure by determining the pharmacodynamic driver for 

efficacy. Each study will contain the following test groups: vehicle control, 

antibiotic alone, ADEP4 alone, antibiotic + ADEP4, and the positive control, ADEP4 + rifampicin (Table 2). 

PKPD driver of ADEP4/antibiotic efficacy. Once an initial efficacious dose of ADEP4 in combination with each 

antibiotic is validated, it will be possible to determine the PD driver for ADEP4 using the methods described by 

Craig (Craig, 2007). Using the neutropenic mouse model described above, groups of 5 mice will be treated for 

1 day with a range of 4 ADEP4 administrations based around the PK and the effective dose from the initial 

efficacy experiment (Table 2). 50 mg/kg ADEP4 sterilized in combination with rifampicin so doses above and 

Treatment # 

Samples 

Mice Route I Route II Total

ADEP4 (25) + Antibiotic 1 8 3 IV IP 6

ADEP4 (50) + Antibiotic 1 8 3 IV IP 6

ADEP4 (100)+Antibiotic 1 8 3 IV IP 6

ADEP4 (25) + Antibiotic 2 8 3 IV IP 6

ADEP4 (50) + Antibiotic 2 8 3 IV IP 6

ADEP4 (100)+Antibiotic 2 8 3 IV IP 6

10% excess 4

Total number of mice 40

Treatment Mice

Vehicle 5

Antibiotic 1 control 5

Antibiotic 2 control 5

Antibiotic 1 + 

ADEP4 

5

Antibiotic 2 + 

ADEP4

5

ADEP4 alone 5

rifampicin + ADEP4 5

10% excess 4

Total 39

Table 2. Initial efficacy study 

design and animal numbers.

Research Strategy Page 71

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
below this dose were selected as an example. ADEP4 doses above the MTD will not be tested, e.g. 4x q24h 

(Table 3). Groups of 5 mice will also be required for untreated, ADEP4 alone and antibiotic alone groups. 

Doses will be fractionated to allow administration of each daily dose q24h, q12h, q8h and q6h to give a broad 

range of AUC, T>MIC and CMax values. The known antibiotic will be administered to test groups using standard 

dosing adjusted for the combined PK similar to the initial efficacy study above. One day after the final dose, 

thigh burden will be determined as previously described. Plotting pathogen count at each dose against AUC, 

T>MIC and CMax will reveal the PK parameter which most closely correlates with efficacy – the PKPD driver. 

Following this design, a total of 105 mice will be required (Table 3).

Treatment Antibiotic

(standard dose) Total

Vehicle N 5

Antibiotic control Y 5

ADEP4 alone (~50 mg/kg) N 5

ADEP4 (0.5x mg/kg q24h) Y 5

ADEP4 (0.5x mg/kg q12h) Y 5

ADEP4 (0.5x mg/kg q8h) Y 5

ADEP4 (0.5x mg/kg q6h) Y 5

ADEP4 (1x mg/kg q24h, q12h, q8h, q6h) Y 20

ADEP4 (2x mg/kg q24h, q12h, q8h, q6h) Y 20

ADEP4 (4x mg/kg q12h, q8h, q6h) Y 15

Uninfected N 5

10% excess N 10

Total number of mice 105

 

Efficacy in implanted biofilms. Combinations which display efficacy in the thigh model will progress to an in vivo

biofilm model (Kristian et al., 2003). In the mouse tissue cage (TC) model, a S. aureus biofilm was not cleared 

with three broad-spectrum antibiotics dosed either alone or in 

combination (Lucet et al., 1990), but we anticipate that our 

combinations will clear the biofilm. Briefly, a sterile TC containing 

sintered glass beads is implanted subcutaneously in the back of the 

anaesthetized mouse. Two weeks after surgery, the TC is verified to 

be sterile, the mice are then immunocompromized, 200 μL of a S. 

aureus culture is introduced into the TC and the animals are left for 

14 days to allow the infection to stabilize. Mice are then dosed for 7 

days and euthanized to allow removal of the TC. Bacterial counts will 

then be performed on the TC fluid and on the glass beads (following 

washing and sonication). Lucet et al. used a fixed dose of each agent 

for their studies and our initial efficacy study will directly compare their 

combination of rifampicin (25 mg/kg) + vancomycin (50 mg/kg) with 

our chosen test combinations. A vehicle control group will also be 

studied. 10 mice per group and 20% excess will be required due to 

the variability of this model and the possibility that once implanted, the 

tissue cages will not remain sterile (Table 4). 

Aim 3. Preclinical development. These studies are generally required by the FDA prior to pre-IND 

discussions. They also provide the basis for a risk-benefit assessment to be completed prior to entry into 

human clinical trials. They will also provide the basis for selecting doses to be tested in humans. Only a single 

partner antibiotic will enter preclinical development. Studies will include: (1) acute and repeated dose toxicity 

studies in 2 species with local irritation assessments; (2) single dose PK studies in 2 species; (3) genotoxicity 

including Ames and chromosomal aberration studies. Preclinical development studies will be out-sourced to a 

contract research organization, which is a standard practice in the Industry. We have chosen (see 

accompanying letter), which will provide cost effective support for the project. 

Table 3. Dose fractionation study design and animal numbers.

Treatment Mice

Vehicle 10

Antibiotic 1 control 10

Antibiotic 2 control 10

Antibiotic 1 + ADEP4 10

Antibiotic 2 + ADEP4 10

ADEP4 alone 10

rifampicin + 

vancomycin 

10

20% excess 14

Total 84

Table 4. Biofilm study design and 

animal numbers.

Research Strategy Page 72

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
 Single dose MTD determination will be performed in 24 Sprague Dawley rats divided into four groups 

(3/sex/group). ADEP4 will be delivered by IV injection in combination with the standard dose of the partner 

antibiotic in an up/down procedure with 2 days between doses. Clinical observations are performed twice daily 

with body weights determined pre-dose and on day 7. Terminal blood draws are performed for clinical 

chemistry and hematology, along with gross necropsy. Organ weights are determined by standard by 

ICH/OECD guidelines and histopathology is performed on lesions as applicable. 

Next, a 7-day repeat dose range-finding study in Sprague Dawley rats will be performed. This study will 

identify acceptable dosing for the 14 day repeat dose GLP toxicity study. The 7-day repeat dose range-finding 

study will be similar to the MTS determination except body weights will be measured daily. Provided reports 

include all observations and dose administration tables for in-life phase, as well as clinical pathology, gross 

observations, and pathology results, as applicable. 

A 14 day repeat dose GLP toxicity study will also be performed using Sprague Dawley rats. Low, 

middle and high doses of ADEP4 will be given along with standard fixed dosing of the partner antibiotic, daily 

for 14 days by IV injection. Local irritation assessments will be performed at the site of injection. Each group 

will contain 10 male and 10 female rats and the study will also include vehicle control groups, bringing the total 

number animals to 80 for this study. Blood sampling will be performed on the first and last day of the dosings 

for analytical dose verifications. Food consumption and body weight will be measured weekly. Opthamalic 

examination will be performed prior to the first dose and during week 2. Clinical pathology will be performed at 

sacrifice and include standard panels include for clinical chemistry, hematology, coagulation, and urinalysis. 

Necropsy will also be performed and tissues will be stored in formalin prior to histopathology. 

Single dose PK studies will also be performed in rats For Sprague Dawley rats, 6 animals will 

be required per group. Doses of ADEP4 will be delivered by IP and IV injection in combination with a standard 

single dosing regimen of the partner antibiotic. ADEP doing will be selected based single dose MTD 

experiments that were previously performed using this species. High and low ADEP doses will be selected for 

each route of administration and 24 animals will be required. Blood plasma will be sampled at 15, 30, 60, 120, 

240, 360, 720 and 1440 minutes. Drug levels will be determined using LC-MS/MS and standard PK 

parameters for each antibiotic will be reported including oral bioavailability, elimination half-life, peak plasma 

concentration (CMax), area under the curve (AUC) and time above MIC (T>MIC). 

Genotoxicity will be tested using the Ames reverse mutation assay in S. typhimurium and E. coli. The 

tests are conducted using strains of Salmonella containing a mutation in the histidine gene and a strain of 

E.coli containing a mutation in the tryptophan gene. In this assay, the auxotrophic strains are exposed to the 

chemical of interest. The cells are plated on auxotrophic minimal media, such as His-, and the number of 

revertants are counted and compared to untreated and mutagenic chemical controls. The number of mutants 

represent the genotoxic potential of the test compound. 6 concentrations of ADEP4 in combination with the 

partner antibiotic will be tested. Chromosomal aberration assays will also be performed with the Chinese 

hamster ovary (CHO) cell line. The cells will be exposed to the ADEP4/antibiotic combination at three 

concentrations. Metaphase cells in the test groups and negative controls will be examined for structural 

chromosomal aberrations using karyotyping. A confirmatory assay will be performed with an extended period 

of exposure in the event of negative results. A similar study will be performed using human lymphocytes. 

 

Pitfalls and alternatives. Antiinfective drug discovery and development are inherently risky. We have 

attempted to mitigate some of these risks by discovering unique properties of a known agent. These 

properties, namely the ability to sterilize biofilm and stationary populations of cells, have the potential to solve a 

large unmet medical need. ADEP4 was previously optimized, but its development was discontinued. It is 

effective in multiple animal models and its target is known and unique. By combining ADEP4 with other 

antibiotics we expect the potential for resistance development to be low.

Research Strategy Page 73

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Project management. Dr. Ken Coleman, PI, will be responsible for the overall oversight of the project, including 

communications between Arietis; our consultants, Steven Leonard and Kim Lewis of Northeastern University; 

 and the NIH. The team will hold monthly conference calls, and yearly meetings in 

Boston to discuss problems and progress of the project. Dr. Coleman has many years of experience leading 

antibiotic drug discovery and development programs at large and small pharmaceutical companies. The end 

result of this project will be a validated lead combination that will enable us to enter into a partnership with a 

Pharmaceutical Company for additional preclinical development studies leading towards an IND, clinical trials, 

marketing and sales of a new therapeutic which targets chronic infections. 

Research Strategy Page 74

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Vertebrate Animal Section 

These studies will be conducted at Boston University Medical Center by Arietis staff under animal 

assurance number following protocols , which were approved by Boston 

University Institutional Animal Use and Care Committee on May 29 , 2013 and January 29th, 2013 

respectively. Additional studies described in the proposal will be outsourced 

1) Provide a detailed description of the use of animals in the work previously outlined in the 

experimental design and methods section. Identify species, strains, ages, sex, and numbers of 

animals to be used.

The aim of the animal studies is to determine pharmacokinetics and efficacy of ADEP4/antibiotic 

combinations following in vitro evaluation. 

We will begin by performing a detailed PK study of ADEP4 in combination with 2 known antibiotics, which will 

inform the dosing for efficacy. For the vast majority of antibacterials, the salient pharmacokinetic driver for

efficacy is the Area Under the Free Plasma Concentration Time Curve (fAUC) and the usual pharmacodynamic 

driver is Minimum Inhibitory Concentration (MIC), with an fAUC/MIC value of 125 usually sufficient for effective 

therapy with suppression of resistant isolates. The plasma pharmacokinetics of ADEP in combination with 2 

antibiotic partners will be determined following dosing by the IV and IP routes. Blood will be withdrawn by 

submandibular bleeds at 15, 30, 45, 60, 90, 270 and 480 minutes post-dose using microsampling and not to 

exceed IACUC blood volume guidelines. At 1440 minutes post-dose, the mice will be euthanized and bled out 

by cardiac puncture. ADEP4 will be dosed at 25 mg/kg, 50 mg/kg and 100 mg/kg to determine whether PK is 

linear. The chosen antibiotic partners will be dosed using the mouse equivalent of the standard human dose. 

Plasma concentrations will be quantified using LC-MS/MS. The PK will inform dosing strategy for subsequent 

efficacy studies. Following this design, 40 mice will be required for the PK study. 6-8 week old female C57BL/6 

mice will be used for the PK study. 

Efficacy. We have used a murine abscess model as a model of recalcitrant infection. The murine 

staphylococcal thigh lesion has been a standard in vivo test for antimicrobials for fifty years or more. In this 

model, an appropriate inoculum is introduced into the thigh muscle of the mouse to form an abscess. The 

infection remains localized for 4 or more days, causing enlargement of the thigh but no systemic infection. In 

most reported studies, chemotherapy commences within 2h of infection, as delaying therapy beyond this point 

usually results in failure to cure. In our hands, using 

immunocompromised mice and delaying therapy until 24h post-infection

results in a highly recalcitrant infection where high dose vancomycin or 

rifampicin dosed twice/day IP for 2 days gives less than a 2-log 

reduction in viable count relative to an untreated control, while a single 

dose combination of rifampicin with ADEP4 cleared the infection. We 

will continue to use this recalcitrant abscess model to evaluate two 

ADEP4/antibiotic combinations. Initial studies will use the level of 

ADEP4 that was effective in the preliminary work (50 μg/ml; 1⁄2 of MTD), 

and the standard mouse adapted human equivalent of the partner 

antibiotic to demonstrate efficacy, and later studies on successful

combinations will focus on optimizing the ratio of the partners and the best dosing regimen for cure. Each 

Treatment # Samples Mice Route I Route II Total

ADEP4 (25) + Antibiotic 1 8 3 IV IP 6

ADEP4 (50) + Antibiotic 1 8 3 IV IP 6

ADEP4 (100)+Antibiotic 1 8 3 IV IP 6

ADEP4 (25) + Antibiotic 2 8 3 IV IP 6

ADEP4 (50) + Antibiotic 2 8 3 IV IP 6

ADEP4 (100)+Antibiotic 2 8 3 IV IP 6

10% excess 4

 Total 40

Treatment Mice

Vehicle 5

Antibiotic 1 control 5

Antibiotic 2 control 5

Antibiotic 1 + ADEP4 5

Antibiotic 2 + ADEP4 5

ADEP4 alone 5

rifampicin + ADEP4 5

10% excess 4

Total 39

Vertebrate Animals Page 75

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
study will contain the following test groups: vehicle control, antibiotic alone, ADEP4 alone, antibiotic + ADEP4,

ADEP4 + rifampicin (positive control). Following this design 39 animals will be required for the initial efficacy 

study. 6-8 week old female ICR/Swiss mice will be used for efficacy studies. 

Next the combinations will progress to an in vivo biofilm model [1]. In a mouse TC model, a staphylococcal 

biofilm is not cleared with three broad-spectrum antibiotics dosed either alone or in combination

[2] but we anticipate that the lead combination from our treatment optimization studies will clear the biofilm in this 

model. Briefly, a sterile TC containing sintered glass beads is implanted

subcutaneously in the back of the anaesthetized mouse. Two weeks 

after surgery, the TC is verified to be sterile, the mice are then 

immunocompromised with cyclophosphamide (150 mg/kg), and 200 μL 

of a S. aureus culture is introduced into the TC and the animals are left 

for 14 days to allow the infection to stabilize. Mice are then dosed for 7 

days and euthanized to allow removal of the TC. Bacterial counts will 

then be performed on the TC fluid and on the glass beads (following

washing and sonication). Lucet et al. used a fixed dose of each agent 

for their studies and our initial efficacy study will directly compare their 

combination of rifampicin (25 mg/kg) + vancomycin (50 mg/kg) with our chosen test combination. An infected 

untreated control group will also be studied. Following this design 84 animals will be required for the tissue cage 

study. 6-8 week old female ICR/Swiss mice will be used for these studies. 

Once an initial efficacious dose of ADEP4 in combination with each antibiotic is validated, it will be possible to 

determine the PD driver for ADEP4 using the methods described by Craig [3]. Using the neutropenic mouse 

model described above, groups of 5 mice will be treated for 1 day with a range of 4 ADEP4 administrations 

based around the PK and the effective dose from the initial efficacy experiment. 50 mg/kg ADEP4 sterilized in 

combination with rifampicin so doses above and below this dose were selected as an example. ADEP4 doses 

above the MTD will not be tested, e.g. 4x q24h. Groups of 5 mice will also be required for untreated, ADEP4

alone and antibiotic alone groups. Doses will be fractionated to allow administration of each daily dose q24h, 

q12h, q8h and q6h to give a broad range of AUC, T>MIC and CMax values. The known antibiotic will be 

administered to test groups using standard dosing adjusted for the combined PK similar to the initial efficacy 

study above. One day after the final dose, thigh burden will be determined as previously described. Plotting 

pathogen count at each dose against AUC, T>MIC and CMax will reveal the PK parameter which most closely 

correlates with efficacy – the PKPD driver. Following this design, a total of 105 mice will be required. 6-8 week 

old female ICR/Swiss mice will be used for these efficacy studies. 

Single dose maximum tolerated dose determination will be performed in 24 Sprague Dawley rats divided into 

four groups (3/sex/group). The rats will be prepubertal to young adult, approximately 7-9 weeks of age and 

approximately 150-400 g at the time of randomization. ADEP4 will be delivered by IV injection in combination 

with the standard dose of the partner antibiotic in an up/down procedure with 2 days between doses. Clinical 

observations are performed twice daily with body weights determined pre-dose and on day 7. Terminal blood 

draws are performed for clinical chemistry and hematology, along with gross necropsy. Organ weights are 

determined by standard by ICH/OECD guidelines and histopathology is performed on lesions as applicable. 

Treatment Mice

Vehicle 10

Antibiotic 1 control 10

Antibiotic 2 control 10

Antibiotic 1 + ADEP4 10

Antibiotic 2 + ADEP4 10

ADEP4 alone 10

rifampicin + vancomycin 10

20% excess 14

Total 84

Treatment Antibiotic

(standard dose)

Total

Vehicle N 5

Antibiotic control Y 5

ADEP4 alone (~50 mg/kg) N 5

ADEP4 (0.5x mg/kg q24h) Y 5

ADEP4 (0.5x mg/kg q12h) Y 5

ADEP4 (0.5x mg/kg q8h) Y 5

ADEP4 (0.5x mg/kg q6h) Y 5

ADEP4 (1x mg/kg q24h, q12h, q8h, q6h) Y 20

ADEP4 (2x mg/kg q24h, q12h, q8h, q6h) Y 20

ADEP4 (4x mg/kg q12h, q8h, q6h) Y 15

Uninfected N 5

10% excess N 10

Total 105

Vertebrate Animals Page 76

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Single dose MTD (acute) in Rats

Sprague Dawley Rats Males Female

dose level 1 3 3

dose level 2 3 3

dose level 3 3 3

dose level 4 3 3

 total N = 24

A 7-day repeat dose range-finding study in Sprague Dawley rats will be performed. The rats will be prepubertal 

to young adult, approximately 7-9 weeks of age and approximately 150-400 g at the time of randomization. This 

study will identify acceptable dosing for the 14 day repeat dose GLP toxicity study. The 7-day repeat dose rangefinding study will be similar to the MTD determination except body weights will be measured daily. Provided 

reports include all observations and dose administration tables for in-life phase, as well as clinical pathology, 

gross observations, and pathology results, as applicable. 

Repeat dose MTD in Rats

Sprague Dawley Rats Males Female

dose level 1 3 3

dose level 2 3 3

dose level 3 3 3

dose level 4 3 3

total N = 24

A 14 day repeat dose GLP toxicity study will also be performed using Sprague Dawley rats. Low, middle and 

high doses of ADEP4 will be given along with standard fixed dosing of the partner antibiotic, daily for 14 days by 

IV injection. The rats will be prepubertal to young adult, approximately 7-9 weeks of age and approximately 150-

400 g at the time of randomization. Local irritation assessments will be performed at the site of injection. Each 

group will contain 10 male and 10 female rats and the study will also include vehicle control groups, bringing the 

total number animals to 80 for this study. Blood sampling will be performed on the first and last day of the 

dosings for analytical dose verifications. Food consumption and body weight will be measured weekly. 

Opthamalic examination will be performed prior to the first dose and during week 2. Clinical pathology will be 

performed at sacrifice and include standard panels include for clinical chemistry, hematology, coagulation, and 

urinalysis. Necropsy will also be performed and tissues will be stored in formalin prior to histopathology. 

Sprague Dawley Rats Males Females 

Control 10 10

Low 10 10

Mid 10 10

High 10 10

total N = 80

Vertebrate Animals Page 77

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Single dose PK studies will also be performed in rats . For Sprague Dawley rats, 6 animals will be 

required per group. Doses of ADEP4 will be delivered by IP and IV injection in combination with a standard 

single dosing regimen of the partner antibiotic. ADEP doing will be selected based single dose MTD experiments 

that were previously performed using this species. High and low ADEP doses will be selected for each route of 

administration and 24 animals will be required. Blood plasma will be sampled at 15, 30, 60, 120, 240, 360, 720 

and 1440 minutes. Drug levels will be determined using LC-MS/MS and standard PK parameters for each 

antibiotic will be reported including oral bioavailability, elimination half-life, peak plasma concentration (CMax), 

area under the curve (AUC) and time above MIC (T>MIC). 

2) Justification for the use of animals.

The use of animals in a drug discovery pipeline is essential for prioritization of lead compounds into 

development candidate drugs for humans. Additionally, animal safety and efficacy data are a regulatory 

requirement in the majority of countries. Many potential drugs fail for PK reasons. Understanding the 

factors that lead to good PK properties is an important part of the design and selection of drug candidates. 

Justification for animal numbers. 

For PK studies, typically an 8 time-point pharmacokinetic profile is generated using 3-6 animals per group. We 

will use 3 mice per group in order to minimize the number of animals used based on similar studies using the 

same model to study drug efficacy and PK testing of approved antibiotics [3, 4]. For example, using three 

animals per group, there was 80% power to detect a difference of 25 ml/min/kg in the clearance of midazolam, 

at a significance level of 0.05 [5]. Three animals per group was also used to calculate the PK profile for a preclinical anti-H. pylori evaluation of the antibiotic finafloxacin [6]. 

Single Dose PK Study in rats

Species Dose

Route

Animals

per group

Doses per

Group

Collection

Time points

Sample

Collection

Sprague

Dawley Rats

IV 6 4 8 time points plasma

N = 24

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
 

For efficacy studies, we will need 5 mice per treatment group in order to produce statistically relevant results. 

The number of mice per group is based on similar studies using the same model to measure antibiotic efficacy, 

which used 5-10 mice per group [7]. We will use the lowest number of mice which has conferred statistical 

significance in this model. Based on variability of this model described in the literature, we expect to detect at 

least a 90% decrease in virulence with a probability of 80% at a two sided 5% significance level using 5 animals 

per group. Since we aim to eradicate infection, we expect our combination therapies to produce more than 90% 

decreased virulence compared to negative controls and individual antibiotics, serving as an excellent indicator 

of efficacy at this stage of development. The Mann-Whitney rank sum test will be used to determine differences 

in the median values of the bacterial count per thigh between antibiotic treated mice and controls. 

For tissue cage studies, we will require 10 mice per group in order to produce statistically relevant results. The 

larger number of mice per group compared to the thigh model is a result of the higher variability of this model. 

Based on variability of this model described in the literature, we expect to detect at least a 99% decrease in 

virulence with a probability of 80% at a two sided 5% significance level using 10 animals per group. As most 

antibiotics are not effective in this model, and detection of a 2-log reduction in CFU with a probability of 80% will 

be sufficient to justify further evaluations for our combined therapeutic. The Mann-Whitney rank sum test will be 

used to determine differences in the median values of the bacterial count per thigh between antibiotic treated 

mice and controls. 

These animal studies will be performed at Boston University Medical Center which is fully accredited by the

Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and operates in full 

compliance with all federal and state regulatory agencies. 

3) Veterinary care of the animals involved.

The animals studies performed by Arietis will be housed in the AAALAC accredited facilities of the Boston

University Medical Campus (BUMC). The animal facilities comprise 

approximately 45,780 sq feet of animal housing and support space. The Laboratory Animal Science Center is a 

team of administrative, managerial, technical and professional staff committed to the advancement of science 

in collaboration with the research community by promoting the humane care and use of animals used in 

biomedical research and teaching. The Laboratory Animal Science Center (LASC) oversees veterinary medical 

care, animal care services, and actively participates in all facility design. Day-to-day care of animals and 

technical assistance are provided by approximately thirty animal caretakers. The care staff is overseen by 

Operations Managers and Animal Care Supervisors. In addition, three veterinary technicians are supervised by 

a Veterinary Services Manager, under the direction of the Attending Veterinarian. Administrative, business and 

purchasing functions are supported by a Business Manager and three support staff. Use of these animal 

facilities is included in the BioSquare lease that Arietis has negotiated with BUMC. Standard Operating 

Procedures and reference materials are available from the IACUC Office for animal use. The animal health 

program for all Boston University owned laboratory animals is directed by the attending veterinarian, 

, and provided by two full-time veterinarians. All studies involving 

animals will be performed in the Laboratory Animal Sciences Center (LASC). Animals in each LASC room are 

observed daily for signs of illness by the animal technician responsible for providing husbandry. Medical 

records and documentation of experimental use are maintained individually for non-rodents and individually or 

by cage group for rodents. Veterinary technicians under the direction of the attending veterinarian provide

routine veterinary medical care to all animals. Animal care and use is additionally monitored for training and 

compliance issues by the Training and Compliance Manager. 

 Staff from Northeastern University and Arietis have previous

experience with the murine thigh abscess model and Arietis has a qualified veterinary surgeon on staff. 

 Page 79

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
 

4) Describe procedures for ensuring that discomfort, distress, pain, and injury will be limited to that 

which is unavoidable to conduct scientifically sound research. Describe the use of analgesic, 

anesthetic, and tranquilizing drugs and comfortable restraining devices, where appropriate, to 

minimize discomfort, distress, pain, and injury.

 For studies performed by Arietis, all IACUC policies on humane endpoints will be followed. Animals will 

be monitored at least every 24 hours for signs of distress or pain. If pain or distress is detected, the choice of 

appropriate analgesic will be made upon consultation with the attending veterinarian. For example, 

pharmaceutical grade carprofen may be given orally at 5 mg/kg every 12-24 h as needed. Sedation is required 

for the manipulation of each mouse prior to thigh injections. Isoflurane will be administered at 1-3% inhalant to 

effect (up to 5% for induction). The animals will not be allowed to reach a moribund state. However if for some 

reason the animals reach a moribund state, they will be immediately euthanized. Animals observed 

experiencing the following humane endpoints will be euthanized: hunched posture,sunken eyes, with or without 

discharge; respiration that has increased, decreased, or appears labored; cyanosis (blue color to skin or 

mucous membranes); hypothermia or hyperthermia; ruffled hair coat, erection of hair or fur, lack of grooming 

behavior; diarrhea or constipation; prolonged bleeding from any orifice; self-mutilation; no response to external 

stimuli; vomiting; unsteady gait or lameness not induced by experimental manipulation; ulcerated tumors; 

severe or ulcerative dermatitis; inability to reach food and/or water; inability to remain upright; refusal of food 

and water monitored through disappearing from bottle and feed container; clinical dehydration and/or prolonged 

decreased food intake (more than 48 hours); muscle atrophy and signs of lethargy and lack of physical activity; 

Decreased body condition score(2.5 or less on a scale of 1-5; mouse in not well conditioned, vertebral column 

may be prominent). The animals will be observed at least once daily during the workdays, and at least once 

daily on holidays and weekends. An animal entering, but not yet in, the moribund state will be euthanized if the 

next observation is going to be after a period longer than 12 hours. 

 

 

Vertebrate Animals Page 80

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
5) Describe any euthanasia method to be used and the reasons for its selection. State whether this 

method is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary 

Medical Association.

For studies performed by Arietis, every animal will be euthanized by CO2 inhalation, with secondary euthanasia 

by cervical dislocation. Methods for euthanasia are consistent with the recommendations of the Panel on 

Euthanasia of the American Veterinary Medical Association and the Boston University animal welfare 

regulations and policies. 

 

References. 

1. Kristian, S.A., et al., Alanylation of teichoic acids protects Staphylococcus aureus against Toll-like receptor 2-

dependent host defense in a mouse tissue cage infection model. J Infect Dis, 2003. 188(3): p. 414-23.

2. Lucet, J.C., et al., Treatment of experimental foreign body infection caused by methicillin-resistant 

Staphylococcus aureus. Antimicrob Agents Chemother, 1990. 34(12): p. 2312-7.

3. Craig, W.A., Pharmacodynamics of antimicrobials: General concepts and applications. 2nd Edition ed. 

Antimicrobial pharmacodynamics in theory and clinical practice., ed. T. Murakawa, Ambrose, PG. 2007, New 

York, New York: Marcel Dekker Inc. 

4. Siefert HM, Kohlsdorfer C, Steinke W et al. Pharmacokinetics of the 8-methoxyquinolone, moxifloxacin: 

tissue distribution in male rats. J Antimicrob Chemother 1999; 43 Suppl B: 61-7.

5. Vallee E, Azoulay-Dupuis E, Pocidalo JJ, Bergogne-Berezin E. Activity and local delivery of azithromycin in a 

mouse model of Haemophilus influenzae lung infection. Antimicrob Agents Chemother 1992; 36: 1412-1417.

6. Yamano K, Yamamoto K, Kotaki H et al. Quantitative prediction of metabolic inhibition of midazolam by 

erythromycin, diltiazem, and verapamil in rats: implication of concentrative uptake of inhibitors into liver. J 

Pharmacol Exp Ther 2000; 292: 1118-26.

7. Endermann R, Ladel C, Stubbings W, Labischinski H. Pharmacokinetics (PK) and In Vivo Efficacy of Oral 

Finafloxacin (FIN) and Comparators in Rodent Models of Systemic Infection. ICAAC 2008, Washington DC, 27th 

October 2008. 

8. Escaich, S., et al., The MUT056399 inhibitor of FabI is a new antistaphylococcal compound. Antimicrob 

Agents Chemother, 2011. 55(10): p. 4692-7.

Vertebrate Animals Page 81

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Allison, K. R., M. P. Brynildsen & J. J. Collins, (2011) Metabolite-enabled eradication of bacterial persisters by 

aminoglycosides. Nature 473: 216-220. 

Balaban, N. Q., J. Merrin, R. Chait, L. Kowalik & S. Leibler, (2004) Bacterial persistence as a phenotypic switch. Science

305: 1622-1625. 

Brotz-Oesterhelt, H., D. Beyer, H. P. Kroll, R. Endermann, C. Ladel, W. Schroeder, B. Hinzen, S. Raddatz, H. Paulsen, K. 

Henninger, J. E. Bandow, H. G. Sahl & H. Labischinski, (2005) Dysregulation of bacterial proteolytic machinery by 

a new class of antibiotics. Nat Med 11: 1082-1087. 

Buske, P. J. & P. A. Levin, (2013) A flexible C-terminal linker is required for proper FtsZ assembly in vitro and cytokinetic 

ring formation in vivo. Molecular microbiology 89: 249-263. 

Conlon, B. P., E. S. Nakayasu, L. E. Fleck, M. D. LaFleur, V. M. Isabella, K. Coleman, S. N. Leonard, R. D. Smith, J. N. Adkins 

& K. Lewis, (2013) Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503: 365-370. 

Craig, W. A., (2007) Pharmacodynamics of antimicrobials: General concepts and applications. Marcel Dekker Inc., New 

York, New York. 

Dorr, T., M. Vulic & K. Lewis, (2010) Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia 

coli. PLoS biology 8: e1000317. 

Dwyer, D. J., M. A. Kohanski & J. J. Collins, (2009) Role of reactive oxygen species in antibiotic action and resistance. 

Current Opinion in Microbiology 12: 482-489. 

Frees, D., S. N. Qazi, P. J. Hill & H. Ingmer, (2003) Alternative roles of ClpX and ClpP in Staphylococcus aureus stress 

tolerance and virulence. Mol Microbiol 48: 1565-1578. 

Gillaspy, A. F., S. G. Hickmon, R. A. Skinner, J. R. Thomas, C. L. Nelson & M. S. Smeltzer, (1995) Role of the accessory gene 

regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infect Immun 63: 3373-3380. 

Hansen, S., K. Lewis & M. Vulić, (2008) The role of global regulators and nucleotide metabolism in antibiotic tolerance in 

Escherichia coli. Antimicrob Agents Chemother. 

Keren, I., N. Kaldalu, A. Spoering, Y. Wang & K. Lewis, (2004a) Persister cells and tolerance to antimicrobials. FEMS 

Microbiol Lett 230: 13-18. 

Keren, I., D. Shah, A. Spoering, N. Kaldalu & K. Lewis, (2004b) Specialized persister cells and the mechanism of multidrug 

tolerance in Escherichia coli. J Bacteriol 186: 8172-8180. 

Keren, I., Y. Wu, J. Inocencio, L. R. Mulcahy & K. Lewis, (2013) Killing by bactericidal antibiotics does not depend on 

reactive oxygen species. Science 339: 1213-1216. 

Kim, J. S., P. Heo, T. J. Yang, K. S. Lee, D. H. Cho, B. T. Kim, J. H. Suh, H. J. Lim, D. Shin, S. K. Kim & D. H. Kweon, (2011) 

Selective killing of bacterial persisters by a single chemical compound without affecting normal antibioticsensitive cells. Antimicrob Agents Chemother 55: 5380-5383. 

Kirstein, J., A. Hoffmann, H. Lilie, R. Schmidt, H. Rubsamen-Waigmann, H. Brotz-Oesterhelt, A. Mogk & K. Turgay, (2009) 

The antibiotic ADEP reprogrammes ClpP, switching it from a regulated to an uncontrolled protease. EMBO Mol 

Med 1: 37-49.

Kohanski, M. A., D. J. Dwyer, B. Hayete, C. A. Lawrence & J. J. Collins, (2007) A common mechanism of cellular death 

induced by bactericidal antibiotics. Cell 130: 797-810. 

Kristian, S. A., X. Lauth, V. Nizet, F. Goetz, B. Neumeister, A. Peschel & R. Landmann, (2003) Alanylation of teichoic acids 

protects Staphylococcus aureus against Toll-like receptor 2-dependent host defense in a mouse tissue cage 

infection model. J Infect Dis 188: 414-423. 

LaFleur, M. D., Q. Qi & K. Lewis, (2010) Patients with long-term oral carriage harbor high-persister mutants of Candida 

albicans. Antimicrob Agents Chemother 54: 39-44.

Lamp, K. C., L. V. Friedrich, L. Mendez-Vigo & R. Russo, (2007) Clinical experience with daptomycin for the treatment of 

patients with osteomyelitis. The American journal of medicine 120: S13-20. 

Lee, B. G., E. Y. Park, K. E. Lee, H. Jeon, K. H. Sung, H. Paulsen, H. Rubsamen-Schaeff, H. Brotz-Oesterhelt & H. K. Song, 

(2010) Structures of ClpP in complex with acyldepsipeptide antibiotics reveal its activation mechanism. Nat 

Struct Mol Biol 17: 471-478. 

Lewis, K., (2010) Persister cells. Annu Rev Microbiol 64: 357-372. 

Liu, Y. & J. A. Imlay, (2013) Cell death from antibiotics without the involvement of reactive oxygen species. Science 339: 

1210-1213. 

Lowth, B. R., J. Kirstein-Miles, T. Saiyed, H. Brotz-Oesterhelt, R. I. Morimoto, K. N. Truscott & D. A. Dougan, (2012) 

Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX. 

Journal of structural biology. 

References Cited Page 82

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Lucet, J. C., M. Herrmann, P. Rohner, R. Auckenthaler, F. A. Waldvogel & D. P. Lew, (1990) Treatment of experimental 

foreign body infection caused by methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 34: 

2312-2317. 

Maisonneuve, E., L. J. Shakespeare, M. G. Jorgensen & K. Gerdes, (2011) Bacterial persistence by RNA endonucleases. 

Proceedings of the National Academy of Sciences of the United States of America 108: 13206-13211. 

Michalik, S., M. Liebeke, D. Zuhlke, M. Lalk, J. Bernhardt, U. Gerth & M. Hecker, (2009) Proteolysis during long-term 

glucose starvation in Staphylococcus aureus COL. Proteomics 9: 4468-4477. 

Michel, K. H. & R. E. Kastner, (1985) A54556 antibiotics and process for production thereof. US patent 4492650. 

Miyazaki, M., T. Takata, H. Yoshimura, A. Matsunaga, D. Ohta, H. Ishikura, M. Futo, S. Hara, H. Kamimura, K. Tamura, D. 

Ngo & B. T. Tsuji, (2011) Vancomycin bactericidal activity as a predictor of 30-day mortality in patients with 

methicillin-resistant Staphylococcus aureus bacteremia. Antimicrobial agents and chemotherapy 55: 1819-1820. 

Mulcahy, L. R., J. L. Burns, S. Lory & K. Lewis, (2010) Emergence of Pseudomonas aeruginosa strains producing high levels 

of persister cells in patients with cystic fibrosis. J Bacteriol 192: 6191-6199. 

Murillo, O., C. Garrigos, M. E. Pachon, G. Euba, R. Verdaguer, C. Cabellos, J. Cabo, F. Gudiol & J. Ariza, (2009) Efficacy of 

high doses of daptomycin versus alternative therapies against experimental foreign-body infection by 

methicillin-resistant Staphylococcus aureus. Antimicrobial agents and chemotherapy 53: 4252-4257. 

Nguyen, D., A. Joshi-Datar, F. Lepine, E. Bauerle, O. Olakanmi, K. Beer, G. McKay, R. Siehnel, J. Schafhauser, Y. Wang, B. 

E. Britigan & P. K. Singh, (2011) Active starvation responses mediate antibiotic tolerance in biofilms and nutrientlimited bacteria. Science 334: 982-986. 

Sass, P., M. Josten, K. Famulla, G. Schiffer, H. G. Sahl, L. Hamoen & H. Brotz-Oesterhelt, (2011) Antibiotic 

acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ. Proc Natl Acad Sci U S A 108: 

17474-17479. 

Schumacher, M. A., K. M. Piro, W. Xu, S. Hansen, K. Lewis & R. G. Brennan, (2009) Molecular mechanisms of HipAmediated multidrug tolerance and its neutralization by HipB. Science 323: 396-401. 

Shah, D., Z. Zhang, A. Khodursky, N. Kaldalu, K. Kurg & K. Lewis, (2006) Persisters: A distinct physiological state of E. coli. 

BMC Microbiol 6: 53-61.

Socha, A. M., N. Y. Tan, K. L. LaPlante & J. K. Sello, (2010) Diversity-oriented synthesis of cyclic acyldepsipeptides leads to 

the discovery of a potent antibacterial agent. Bioorganic & medicinal chemistry 18: 7193-7202. 

Stryjewski, M. E. & H. F. Chambers, (2008) Skin and soft-tissue infections caused by community-acquired methicillinresistant Staphylococcus aureus. Clin Infect Dis 46 Suppl 5: S368-377. 

References Cited Page 83

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
School of 

Pharmacy

Department of 

Pharmacy Practice

360 Huntington Ave., 

R218TF

Boston, MA 02115

August 1, 2013

Dr. Ken Coleman

Arietis Corporation

650 Albany St, 

Boston, MA 02118 

Dear Ken,

I am happy to consult on your project, “Antibiotics for Recalcitrant Infection.” As you 

know, I specialize in pharmacology and specifically using the hollow-fiber model to test the 

effectiveness of antibiotics against S. aureus. It is clear from the hollow-fiber study that 

ADEP4 has exceptional killing properties and I was not surprised that these results 

translated nicely into animal efficacy. In my opinion, the resistance studies that you propose 

are well designed and will also be predictive whether resistance development for your 

combined therapeutic will be a concern. Of course, these experiments will also be useful in 

prioritizing which antibiotic partner to choose. In addition to the hollow-fiber model, I can 

assist on many other aspects of the proposal including PK/PD and dosing for in vivo efficacy 

studies. I wish you luck with this important application and I look forward to continuing to

work on this exciting project together.

Sincerely,

Steven N. Leonard, Pharm.D.

Assistant Professor, Department of Pharmacy Practice

Northeastern University School of Pharmacy

Clinical Pharmacist, Infectious Diseases

Brigham and Women’s Hospital

 

Letters of Support Page 84

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
Northeastern University

Kenneth Coleman, Ph.D. 8/1/201 

 

Arietis Corporation

650 Albany St., 

Boston, MA 02118

Dear Ken,

I will be happy to continue our collaboration on developing a sterilizing therapeutic 

based on ADEP, and act as a consultant for your SBIR project. As you know, the 

focus of my lab is on mechanisms of antimicrobial drug tolerance and persister cells. 

What we learned over the years is that the mechanisms that form drug-tolerant 

persisters are redundant, and no realistic drug target emerged from our studies. The 

only thing that would sterilize a stationary or biofilm population of cells in our hands 

was peracetic acid. The finding that the ClpP activator, ADEP forces cells to selfdigest and sterilizes a mouse deep-seated infection in combination with rifampicin is 

truly exciting. I think we finally have a good candidate to address untreatable chronic 

infections. 

Best wishes,

Kim Lewis 

University Distinguished Professor 

Director,

Antimicrobial Discovery Center

134 Mugar

360 Huntington Avenue

Boston, MA 02115

www.biology.neu.edu/

faculty03/lewis03.html

Letters of Support Page 85

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
We will disseminate the results of this study by publishing it in professional Journals such as Antimicrobial 

Agents and Chemotherapy, and by presenting at professional meetings (ICAAC). 

 

Resource Sharing Plan Page 87

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
PHS 398 Checklist

OMB Number: 0925-0001 

1. Application Type:

From SF 424 (R&R) Cover Page. The responses provided on the R&R cover page are repeated here for your reference, as you answer 

the questions that are specific to the PHS398.

* Type of Application:

Federal Identifier: 

2. Change of Investigator / Change of Institution Questions

Change of principal investigator / program director

Name of former principal investigator / program director: 

Change of Grantee Institution

* Name of former institution:

3. Inventions and Patents (For renewal applications only)

* Inventions and Patents:

If the answer is "Yes" then please answer the following:

* Previously Reported: 

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* Last Name:

Suffix:

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Yes No

	


 		

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Checklist Page 88

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth
4. * Program Income

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and 

source(s). Otherwise, leave this section blank.

Is program income anticipated during the periods for which the grant support is requested?

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5. * Disclosure Permission Statement

If this application does not result in an award, is the Government permitted to disclose the title of your proposed project, and the name, 

address, telephone number and e-mail address of the official signing for the applicant organization, to organizations that may be 

interested in contacting you for further information (e.g., possible collaborations, investment)? 

Yes No

	


 		

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Checklist Page 89

Principal Investigator/Program Director (Last, first, middle): Coleman, Kenneth