Document:

Exhibit 10.23 - AMENDED WELLS FARGO CREDIT AGREEMENT

		

			Exhibit 10.23

		

		
			FIRST AMENDMENT TO CREDIT AGREEMENT 
		

		
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			THIS FIRST AMENDMENT TO CREDIT AGREEMENT (this "Amendment") is entered into as of April 22, 2022, by and among the lenders identified on the signature pages hereof (such lenders, together with their respective successors and permitted assigns, are referred to hereinafter each individually as a "Lender" and collectively as "Lenders"), WELLS FARGO BANK, NATIONAL ASSOCIATION, a national banking association, as administrative agent for each member of the Lender Group and the Bank Product Providers (in such capacity, together with its successors and assigns in such capacity, "Agent"), and FLEXSTEEL INDUSTRIES, INC., a Minnesota corporation ("Borrower").
		

		
			WHEREAS, the Borrower, Agent, and Lenders are parties to that certain Credit Agreement dated as of September 8, 2021 (as amended, restated, supplemented or otherwise modified from time to time, the "Credit Agreement"); 
		

		
			WHEREAS, the Borrower has requested that the Lenders effect certain amendments to the Credit Agreement; and
		

		
			WHEREAS, the Agent and the undersigned Lenders have agreed to effect such amendments to the Credit Agreement, in each case subject to the terms and provisions hereof;
		

		
			NOW THEREFORE, in consideration of the premises and mutual agreements herein contained, the parties hereto agree as follows:
		

			
	
			
				 1.
			

			
	
			
			Defined Terms.  Unless otherwise defined herein, capitalized terms used herein and not otherwise defined shall have the meanings ascribed to such terms in the Credit Agreement.

			
	
			
				 2.
			

			
	
			
			Amendment. Subject to the satisfaction of the conditions precedent set forth in Section 5 below and in reliance upon the representations and warranties of the Borrowers set forth in Section 6 below, the definition of "Payment Conditions" set forth in Section 1.1 of the Credit Agreement is hereby amended and restated in its entirety as follows:.

		
			"Payment Conditions" means, at the time of determination with respect to a proposed payment to fund a Specified Transaction, that:
		

		
			(a)no Default or Event of Default then exists or would arise as a result of the consummation of such Specified Transaction, and
		

		
			(b)either
		

		
			(i) both (A) the Fixed Charge Coverage Ratio of the Loan Parties and their Subsidiaries is equal to or greater than 1.10:1.00 for the trailing 12 month period most recently ended for which financial statements are required to have been delivered to Agent pursuant to Schedule 5.1 to this Agreement (calculated on a pro 
		

		 

 

		forma basis as if such proposed payment is a Fixed Charge made on the last day of such 12 month period (it being understood that such proposed payment shall also be a Fixed Charge made on the last day of such 12 month period for purposes of calculating the Fixed Charge Coverage Ratio under this clause (b) for any subsequent proposed payment to fund a Specific Transaction)), and (B) Availability, (x) at all times during the 60 consecutive days immediately preceding the date of such proposed payment and the consummation of such Specified Transaction, calculated on a pro forma basis as if such proposed payment was made, and the Specified Transaction was consummated, on the first day of such period, and (y) after giving effect to such proposed payment and Specified Transaction, in each case, is not less than 15% of the Maximum Revolver Amount, 
		

		
			or
		

		
			(ii)Availability, (x) at all times during the 60 consecutive days immediately preceding the date of such proposed payment and the consummation of such Specified Transaction, calculated on a pro forma basis as if such proposed payment was made, and the Specified Transaction was consummated, on the first day of such period, and (y) after giving effect to such proposed payment and Specified Transaction, in each case, is not less than 17.5% of the Maximum Revolver Amount, and
		

		
			(c)with respect to any proposed payment in an amount equal to or in excess of $1,500,000, Administrative Borrower has delivered a certificate or notification to Agent (which certificate or notification may be delivered to Agent by electronic mail) certifying that all conditions described in clauses (a) and (b) above have been satisfied.
		

			
	
			
				 3.
			

			
	
			
			Continuing Effect.  On and after the Amendment Effective Date, references in the Credit Agreement to "this Agreement" (and indirect references such as "hereunder", "hereby", "herein", and "hereof") and in any Loan Document to the "Credit Agreement" shall be deemed to be references to the Credit Agreement as modified hereby.  Except as expressly set forth in Section 2 of this Amendment, nothing in this Amendment shall constitute a modification or alteration of the terms, conditions or covenants of the Credit Agreement or any other Loan Document, or a waiver of any other terms or provisions thereof, and the Credit Agreement and the other Loan Documents shall remain unchanged and shall continue in full force and effect, in each case as modified hereby.  This Amendment is a Loan Document.

			
	
			
				 4.
			

			
	
			
			Reaffirmation and Confirmation.  Borrower hereby ratifies, affirms, acknowledges and agrees that the Credit Agreement and the other Loan Documents to which it is a party represent its valid, enforceable and collectible obligations, and further acknowledges that there are no existing claims, defenses, personal or otherwise, or rights of setoff whatsoever with respect to the Credit Agreement or any other Loan Document.  Borrower hereby agrees that this Amendment in no way acts as a release or relinquishment of the Liens and rights securing payments of the Obligations.  The 
		

		 

		

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			Liens and rights securing payment of the Obligations are hereby ratified and confirmed by Borrower in all respects.

			
	
			
				 5.
			

			
	
			
			Conditions to Effectiveness.  The effectiveness of this Amendment is expressly conditioned upon the satisfaction of each of the following conditions precedent in a manner satisfactory to each Agent (the date on which such conditions have been satisfied, the “Amendment Effective Date”):

			
	
			
				 (a)
			Agent shall have received a copy of this Amendment, executed and delivered by (i) Lenders, and (ii) the Borrower; 

			
	
			
				 (b)
			The representations and warranties of the Borrower set forth in Section 6 below shall be true and correct as of the date hereof; and

			
	
			
				 (c)
			No Default or Event of Default shall have occurred and be continuing.

			
	
			
				 6.
			

			
	
			
			Representations and Warranties.  In order to induce Agent and Required Lenders to enter into this Amendment, Borrower hereby represents and warrants to Agent and Lenders, after giving effect to this Amendment:

			
	
			
				 (a)
			Each of the representations and warranties of Borrower or its Subsidiaries contained in the Credit Agreement or in the other Loan Documents shall be true and correct in all material respects (except that such materiality qualifier shall not be applicable to any representations and warranties that already are qualified or modified by materiality in the text thereof) on and as of the date hereof (except to the extent that such representations and warranties relate solely to an earlier date, in which case such representations and warranties shall be true and correct in all material respects (except that such materiality qualifier shall not be applicable to any representations and warranties that already are qualified or modified by materiality in the text thereof) as of such earlier date);

			
	
			
				 (b)
			No Default or Event of Default has occurred and is continuing; and

			
	
			
				 (c)
			This Amendment and the Credit Agreement, as amended hereby, constitute legal, valid and binding obligations of Borrower and are enforceable against Borrower in accordance with their respective terms, except as enforcement may be limited by equitable principles or by bankruptcy, insolvency, reorganization, moratorium, or similar laws relating to or limiting creditors' rights generally.

			
	
			
				 7.
			

			
	
			
			Miscellaneous.

			
	
			
				 (a)
			Expenses.  Borrower agrees to pay on demand all Lender Group Expenses of Agent and Lenders in connection with the preparation, negotiation, execution, delivery and administration of this Amendment in accordance with the terms of the Credit Agreement.

			
	
			
				 (b)
			Governing Law.  This Amendment shall be a contract made under and governed by, and construed in accordance with the internal laws of the State of Illinois.

		 

		

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				 (c)
			Counterparts.  This Amendment may be executed in any number of counterparts, and by the parties hereto on the same or separate counterparts, and each such counterpart, when executed and delivered, shall be deemed to be an original, but all such counterparts shall together constitute but one and the same Amendment.  Delivery of an executed signature page of this Amendment by facsimile transmission or electronic photocopy (i.e. "pdf") shall be effective as delivery of a manually executed counterpart hereof.

			
	
			
				 8.
			

			
	
			
			Release.  In consideration of the agreements of Agent and Lenders contained herein and for other good and valuable consideration, the receipt and sufficiency of which is hereby acknowledged, Borrower, on behalf of itself and its respective successors and assigns, hereby absolutely, unconditionally and irrevocably releases, remises and forever discharges Agent and Lenders, and their successors and assigns, and their present and former shareholders, affiliates, subsidiaries, divisions, predecessors, directors, officers, attorneys, employees, agents and other representatives (Agent, each Lender and all such other Persons being hereinafter referred to collectively as the "Releasees" and individually as a "Releasee"), of and from all demands, actions, causes of action, suits, covenants, contracts, controversies, agreements, promises, sums of money, accounts, bills, reckonings, damages and any and all other claims, counterclaims, defenses, rights of set‐off, demands and liabilities whatsoever (individually, a "Claim" and collectively, "Claims") of every name and nature, known as of the date of this Amendment, both at law and in equity, which Borrower, or any of its respective successors or assigns may now or hereafter own, hold, have or claim to have against the Releasees or any of them for, upon, or by reason of any circumstance, action, cause or thing whatsoever which arises at any time on or prior to the day and date of this Amendment, in each case for or on account of, or in relation to, or in any way in connection with any of the Credit Agreement, or any of the other Loan Documents or transactions thereunder or related thereto, except with respect to any Claim against any Releasee not known to Borrower on the date hereof that a court of competent jurisdiction finally determines to have resulted from the gross negligence or willful misconduct, or a material breach of the obligations under this Amendment or any of the other Loan Documents, of such Releasee or its officers, directors, employees, attorneys or agents. 

		
			[Signature pages follow]
		

		
			 
		

		

		

		 

		

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		IN WITNESS WHEREOF, the parties hereto have caused this Amendment to be executed by their respective officers thereunto duly authorized and delivered as of the date first above written.
		

		
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						BORROWER:

					
					
						FLEXSTEEL INDUSTRIES, INC.
a Minnesota corporation

By:/s/ Derek P. Schmidt 4/22/2022
Name:  Derek P. Schmidt
Title:   Chief Financial Officer & Chief Operating Officer

				

		
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						WELLS FARGO BANK, NATIONAL ASSOCIATION, a national banking association, as Agent and as a Lender

By:
/s/ Pamela M. Solares
Name: Pamela M. Solares
Title: Vice President

				

		
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			Signature Page to First Amendment to Credit AgreementExhibit 10.30

 

RESEARCH
AGREEMENT

(Non-Clinical)

 

This
Agreement (“Agreement”) entered into this 1st day of July, 2022 (“Effective Date”), by and
between the University of Miami, (“University”) and Jupiter Neurosciences, Inc. (“Company”) to conduct
a study entitled: EVALUATION of JOTROL in PARKINSON’S DISEASE MODELS (“Study”) as described in the protocol/scope
of work attached hereto as Exhibit A and made a part hereof.

 

1.
Principal Investigator.

 

The
Principal Investigator(s) for University shall be Shaun Brothers. The Technical Director(s) for Company shall be Alison D. Silva.

 

2.
Amount of Funding.

 

The
amount of the funding for the conduct of the Study is $72,844.00 (Exhibit B). One-Hundred percent (100%) of this amount will be paid
by Company to University. $24,000 to be paid upon execution of this Agreement, $24,000 upon first dosing of JOTROL in the animals and
the remaining amount, $24,844 upon completion of final study result report. Amendments to the original agreement must be paid in full
(100%) upon execution.

 

	 	All payments shall be paid to the
	 	 	 
	 	          University of Miami (Tax I.D. # 59-0624458)
	 	          and sent to:
	 	          Office of Research Administration
	 	          PO Box 405803
	 	          Atlanta, GA 30384-5803
	 	 	 
	 	Also
    mail a copy to:	Shaun
    Brothers
	 	 	1501
    NW 10th Ave, Room 416
	 	 	Miami,
    Florida 33136-1012

 

It
is expected that grant funds will be expended in general accordance with the budget attached. Actual expenditures may vary at the discretion
of University. Upon completion of the Study, any unexpended funds will be retained by University.

 

3.
Reporting Requirements (if any).

 

A
study report will be sent to the Company after completion and receipt of the final payment.

 

4.
Term of Agreement.

 

Performance
of this Agreement shall begin upon the Effective Date and shall continue for a period of one (1) year unless earlier terminated pursuant
to this Agreement. Either Company or University may terminate this Agreement upon thirty (30) days written notice for any reason.

 

    	 

    	 

    

 

In
the event of such termination, both Company and the University shall take all reasonable steps to cancel further costs in connection
with this agreement. Company and University will be entitled to reimbursement for costs and non-cancelable obligations incurred prior
to effective day of the termination, except in no event shall such reimbursement exceed the total amount stipulated in Section 2.

 

5.
Confidential Information.

 

The
parties may disclose to each other certain confidential and proprietary information and materials relating to the Study, or other proprietary
information of a technical, business or other nature for the purpose of facilitating, supporting and/or conducting the Study. All confidential
and proprietary information exchanged by the parties shall constitute “Confidential Information.”

 

Each
party agrees that, for a period of five (5) years following the termination or expiration of this Agreement, it shall retain in confidence
the Confidential Information of the other party, and will prevent disclosure of such Confidential Information to third parties. Such
restriction shall not apply to Confidential Information which:

 

	 	a)	was
    available to the general public or otherwise part of the public domain prior to or at the time of disclosure to the receiving party;
	 	b)	became
    generally available to the public or otherwise part of the public domain after its disclosure to the receiving party other than through
    an act or omission of the receiving party;
	 	c)	was
    already properly known to the receiving party at the time of disclosure to the receiving party as evidenced by prior written records
    of the receiving party;
	 	d)	was
    properly disclosed to the receiving party, other than under an obligation of confidentiality, by a third party who had no obligation
    of confidentiality to the disclosing party not to disclose such information to others;
	 	e)	was
    published pursuant to Section 6 of this Agreement; or
	 	f)	was
    independently developed by employees of the receiving party without reference to the disclosing party’s Confidential Information.

 

6.
Publication Rights.

 

University
shall have publication privileges in reference to the Study. Company agrees that the Principal Investigator for the University shall
be permitted to publish in journals, theses, dissertations, or other formats of their own choosing, and to present at symposia and national
or regional professional meetings, the methods and results of the Study, subject to the following. At least thirty (30) days in advance
of the submission of such proposed publication or presentation to a journal, editor, or other third party, University shall furnish Company
copies of the proposed publication, abstract, theses, dissertations or presentation. The purposes for such prior submission are: (i)
to provide Company with the opportunity to review and comment on the contents of the proposed publication or presentation; (ii) to identify
any Confidential Information to be deleted from the proposed publication or presentation (excluding Study data and results); and (iii)
to allow time for any patentable subject matter to be identified. Company shall provide any comments to University within thirty (30)
days of receipt of the proposed publication or presentation. University shall give due consideration to any comments made by Company
however, University shall have no obligation to incorporate Company’s comments into the publication. University hereby agrees to
delete from the proposed publication any Confidential Information which Company requests University to delete but only to the extent
such deletion does not preclude the complete and accurate presentation and interpretation of the Study results. Company shall have thirty
(30) days after receipt of the proposed publication or presentation to object to the proposed publication or presentation on the grounds
that there is patentable subject matter that needs protection. In the event Company makes such objection, the University shall refrain
from making such publication or presentation for no longer than sixty (60) days from the date of receipt of such objection (unless extended
by written agreement of the Parties) in order for patent application(s) directed to the patentable subject matter contained in the proposed
publication or presentation to be filed.

 

    	2

    	 

    

 

In
any publication in connection with the Study, University shall acknowledge the contributions of Company as scientifically appropriate.

 

University
shall furnish all data resulting from this Study to the Company.

 

7.
Intellectual Property.

 

Ownership
of inventions, discoveries, works of authorship and other developments existing as of the Effective Date hereof, and all patents, copyrights,
Confidential Information, trade secret rights and other intellectual property rights therein (collectively, “Pre-existing Intellectual
Property”), is not affected by this Agreement, and neither party shall have any claims to or rights in any Pre-existing Intellectual
Property of the other party, except as may be otherwise expressly provided in any other written agreement between them.

 

Title
to any inventions or discoveries conceived or reduced to practice by Company pursuant to conducting the Study shall belong to Company
(“Company Inventions”). Title to any inventions or discoveries conceived or reduced to practice by University that are related
to or arise out of the Study or any improvements made by University upon materials provided by Company for the conduct of the Study shall
be owned jointly by both Company and the University (“Joint Inventions”). Title to any other inventions or discoveries conceived
or reduced to practice by University pursuant to conducting the Study shall belong to University (“University Inventions). University
shall retain, at all times, a non-exclusive worldwide royalty free license to use Company Inventions and Company’s interest in
Joint Inventions to perform the Study, for its internal educational, non-commercial research, and patient care purposes, and to comply
with any applicable laws and regulations.

 

8.
Indemnification.

 

Each
party shall be solely responsible for the payment of any and all claims for loss, personal injury, death, property damage, or otherwise
arising out of any act or omission of its employees or agents in connection with the performance of its obligations under this Agreement.
Each party agrees to indemnify and hold the other party harmless from any and all claims, loss, damages, costs, expenses, actions, lawsuits
and judgments thereon including attorneys’ and experts’ fees and costs through the appellate levels (the “Indemnifying
Party”) made against or incurred by the other party (the “Indemnified Party”) arising out of or relating to any act
or omission of the Indemnifying Party, its agents and employees.

 

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The
provisions of this paragraph and paragraph 8 shall continue after the termination of this Agreement.

 

9.
Insurance.

 

Company
agrees to carry and keep in force, at its expense, general liability insurance with limits not less than $1,000,000 per occurrence and
$2,000,000 aggregate to cover liability for damages on account of bodily or personal injury or death to any person, or damage to property.

 

Prior
to execution of this Agreement, Company shall provide a certificate of insurance or a self-insurance letter (if Company is self-insured)
stating the limits of coverage.

 

10.
University Employees

 

Unless
otherwise approved in writing by University, only University employees shall participate in any professional and technical activities
of this Study. Company agrees to release, hold harmless and indemnify University from and against any and all losses, claims, or damages,
including bodily injury or death or property damage, and including attorney fees through the appellate level, suffered by Company, and
its agents and employees while on University premises, or arising out of or relating to any act or omission of Company, its agents and
employees, provided that such loss, claim or damage does not arise out of negligence of University.

 

11.
Use of Name.

 

Company
agrees that it will not under any circumstances use the name of University or any faculty or employee in advertising, press release,
publicity, or other public announcement, written or verbal, whether to the public press or otherwise, without the express written permission
of University Assistant Vice President for Business Services, Humberto Speziani. University shall acknowledge Company’s support
of the research program under this Agreement in scientific publications and other scientific communications.

 

12.
Materials.

 

All
equipment and materials acquired for use in connection with the Study will be the property of University at the termination of the Agreement.
Except as may be otherwise provided in this Agreement, title to any equipment provided by Company to University shall pass to University
at the time of delivery thereof to University. Any assays, sequences, clones, mutants, or technologies not directly related to the Company’s
technology, developed or discovered by the investigators shall be the property of the investigators in accordance with University of
Miami policies.

 

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13.
WARRANTIES.

 

UNIVERSITY
MAKES NO WARRANTIES, EXPRESS OR IMPLIED AND HEREBY DISCLAIMS ALL SUCH WARRANTIES, AS TO ANY MATTER WHATSOEVER INCLUDING, WITHOUT LIMITATION,
THE CONDITION OF THE RESEARCH OR ANY INVENTION(S) OR PRODUCT(S), WHETHER TANGIBLE OR INTANGIBLE, CONCEIVED, DISCOVERED, OR DEVELOPED
UNDER THIS AGREEMENT; OR THE OWNERSHIP, MERCHANTABILITY, OR FITNESS FOR A PARTICULAR PURPOSE OF THE RESEARCH OR ANY SUCH INVENTION OR
PRODUCT. UNIVERSITY SHALL NOT BE LIABLE FOR ANY DIRECT, CONSEQUENTIAL, OR OTHER DAMAGES SUFFERED BY ANY LICENSEE OR ANY THIRD PARTIES
RESULTING FROM THE USE OF THE RESEARCH OR ANY SUCH INVENTION OR PRODUCT.

 

The
provisions of this paragraph shall continue beyond the termination of this Agreement.

 

14.
Federal Regulations.

 

No
human subject testing shall be conducted under this Agreement. Any studies involving the use of vertebrate animals shall comply with
all state and federal statutes, rules and regulations governing animal care and use. Any studies involving isotopes must comply with
any and all applicable state and federal rules, regulations and statutes. Recombinant DNA research shall be performed in accordance with
regulations promulgated as Guidelines for Research Involving Recombinant DNA Molecules, U.S. Department of Health and Human Services,
Public Health Service, National Institutes of Health.

 

15.
Conflicts.

 

In
the event there is a conflict with the terms of this Agreement, the protocol/scope of work or any other documents pertaining to this
Study, the terms of this Agreement shall govern.

 

16.
Governing Law.

 

This
Agreement shall be governed by the laws of the State of Florida.

 

17.
Notices.

 

All
notices which either party is required to give to the other in conjunction with this Agreement shall be in writing, and shall be given
by certified mail, return receipt requested, or by delivering the same personally, or by courier or Federal Express (or comparable overnight
courier) to such other party, or by facsimile (with confirmation by any other method accepted herein). Any notice given by certified
mail shall be deemed to have been received three (3) days following the date of mailing. If hand delivered or delivered by same day or
overnight courier or by facsimile, such notice shall be deemed to have been received on the date of delivery to the party being noticed.
All notices shall be sent to the addresses specified below:

 

	University
    of Miami

    Attn:
    Exec. Director, Office of Research

    Administration

    1320
    S. Dixie Highway,

    Gables
    One Tower, #650

    Coral
    Gables, FL 33146-1320

    Email:
    CRIS@med.miami.edu

    cc:

    Shaun
    Brothers

    1501
    NW 10th Ave

    Room
    416

    Miami,
    Florida 33136-1012

    Email:
    sbrothers@med.miami.edu
	Jupiter
    Neurosciences, Inc.

    Attn:
    Christer Rosén

    1001
    N US HWY 1, Suite 504

    Jupiter,
    FL 33477

    Email:
    c.rosen@jupiterneurosciences.com

     

    cc:
    Alison Silva

    Email:
    a.silva@jupiterneurosciences.com

     

    cc:
    Marshall hayward

    Email:
    m.hayward@jupiterneurosciences.com

 

18.
Entire Agreement.

 

There
are no oral agreements with respect to the subject matter of this Agreement which are not fully expressed herein. No representations,
understanding, or agreements have been made or relied upon in the making of this Agreement other than those specifically set forth herein.
This Agreement can only be modified by a writing signed by duly authorized representatives of both parties.

 

[Remainder
of page left intentionally blank]

 

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IN
WITNESS THEREOF, the parties have executed this agreement by their duly authorized officers on the date first herein set out:

 

	UNIVERSITY
    OF MIAMI:	 	COMPANY:
	 	 	 
	/s/
    Brandon Stickland, JD	 	/s/
    Christer Rosén
	Authorized
    Organizational Representative	 	Authorized
    Company Representative
	 	 	 
	Brandon
    Strickland, JD,	 	Christer
    Rosén
	Exec.
    Director, Office of Research Admin	 	Chairman
    and CEO
	Name
    and Title	 	Name
    and Title
	 	 	 
	June
    21, 2022	 	June
    21, 2022
	Date	 	Date

 

    	6

    	 

    

 

EXHIBIT
A

Protocol/Scope
of Work

 

    	 

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

 

 

EVALUATION
of JOTROL

 

in
PARKINSON’S DISEASE MODELS

 

 

 

 

Shaun
Brothers, PhD, MBA

Candace H. Carriere, PhD

 

 

 

 

 

 

 

 

    	Confidential	1

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

PROPOSAL
OVERVIEW

 

The
investigations proposed here aim to first, validate JOTROL as a preclinical candidate for the treatment of Parkinson’s disease
(PD). Second, to determine that this drug represents the best possible candidate as compared to currently available treatment options.
To validate other candidates in the same drug series, such as unformulated resveratrol and piceatannol for response and efficacy. To
begin to work out the mechanism of action for this compound to alleviate motor dysfunction. To validate the mechanism of this drug in
animals and animal models of PD. To determine whether the high blood concentrations of JOTROL will be meaningfully achieved and how this
translates to its therapeutic efficacy in this disease state, similarly to its potential clinical relevance for other neurological diseases
as Alzheimer’s disease and MPSI. Finally, we aim to begin the data gathering process that is necessary to enable human experimentation
and clinical trials. Particularly for this, the experimental approach was modeled on the identical ways in which current clinical candidates
for PD were validated for having human therapeutic potential.

 

PROJECT
GOAL

 

Overall
Goal: Evaluate JOTROL activity in a classic toxin model of Parkinson’s disease in mice.

 

Objective
1: Validate the protective capacity of JOTROL in a unilateral intracranial MPTP model using motor behavioral testing.

 

	 	●	The
    neuroprotective capacity of JOTROL will be evaluated on general health and motor behaviors of animals unilaterally microinfused with
    MPTP. Well validated PD behavioral tests will be utilized following the infusion of either vehicle or toxin, including open-field,
    grip strength, rotarod, L-DOPA responsivity and methamphetamine-induced rotations, while also monitoring the animals’ weights
    throughout the duration of the study.

 

Objective
2: Validate protective capacity of JOTROL on dopaminergic neurons in the SN and dopamine content in the striatum.

 

	 	●	The
    neuroprotective actions of JOTROL will be determined by assessing altered tyrosine hydroxylase (TH) activity in the SN, TH, dopamine
    transporter (DAT), dopamine and GABA content in the striatum and other analyses of basal ganglia neuronal populations involved in
    PD and motor function. This will be conducted in the unilateral MPTP model of PD and control animals that were treated with JOTROL.

 

    	Confidential	2

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

PROPOSED
PROJECT SCHEDULE

 

	TIMELINE	 	PROPOSED
    TREATMENT	 	PROPOSED
    WORK
	 	 	 	 	Baseline Behavior
	WEEK
    1	 	None	 	Weights

    Open-Field
    Test

	 	 	 	 	Grip Strength
	 	 	 	 	Rotarod
	 	 	 	 	 
	 	 	 	 	Stereotaxic Surgery
	 	 	Unilateral Infusion with
    either Vehicle or MPTP into the SN and MFB (50 μg)	 	C57/BL6
                                   Mice (30+ grams), N=8/group (Total: 40 mice)

                                   5
                                   Groups:

	WEEK
    2	 	Daily
    oral gavage of Vehicle or JOTROL starting same day of surgery (first dose immediately after surgery completion)	 	

    Sham-infused,
    Vehicle-treated

    Sham-infused,
    JOTROL-treated

    MPTP-infused,
    Vehicle-treated 

    MPTP-infused,
    JOTROL-treated (Low Dose: 25 mg/kg)

    MPTP-infused,
    JOTROL-treated (High Dose: 50 mg/kg)

	 	 	 	 	 
	WEEK
    3	 	Daily
    oral gavage of Vehicle or JOTROL	 	Recovery
    Week – remove wound clips 7-10 days post-op

    Weights

	 	 	 	 	 
	

    WEEK 4
	 	Daily
    oral gavage of Vehicle or JOTROL	 	Weights

    Open-Field
    Test

    Grip
    Strength

    Rotarod

	 	 	 	 	 
	WEEK 5
	 	Daily
    oral gavage of Vehicle or JOTROL	 	Weights

    Open-Field
    Test

    Grip
    Strength

    Rotarod

	 	 	 	 	 
	 

    WEEK 6
	 	Daily
    oral gavage of Vehicle or JOTROL	 	Weights

    Open-Field
    Test

    Grip
    Strength

    Rotarod

	 	 	 	 	 
	

    Week 7
	 	Daily
    oral gavage of Vehicle or JOTROL	 	Weights

    Open-Field
    Test

    Grip
    Strength

    Rotarod

	 	 	 	 	 
	Week
    8	 	Daily
    oral gavage of Vehicle or JOTROL	 	Weights

    L-DOPA
    Responsivity: Grip Strength & Rotarod

	 	 	 	 	 
	

    Week 9
	 	Daily oral gavage
    of Vehicle or JOTROL 

    Methamphetamine,
    i.p. (5 mg/kg)
	 	Weights

    Methamphetamine-induced
    Rotations

	 	 	 	 	 
	Week
    10	 	Daily
    oral gavage of Vehicle or JOTROL	 	Weights

    Open-Field
    Test

    Grip
    Strength

    Rotarod

	 	 	 	 	 
	Week
    11	 	Daily
    oral gavage of Vehicle or JOTROL	 	Transcardial Perfusions
    or Fresh Tissue Collection
	 	 	 	 	 
	Week
    12+	 	N/A	 	Western Blot & HPLC
    Analysis of Brain Samples

 

    	Confidential	3

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

BACKGROUND

 

Parkinson’s
disease (PD), the second most common age-related neurodegenerative disorder after Alzheimer’s disease1,2, is characterized
by the loss of dopamine-producing neurons within the substantia nigra pars compacta (SN) of the direct motor pathway, which in turn contributes
to postsynaptic dopamine depletion within the striatum. This loss of dopamine functionality results in motor behavioral deficits including
uncontrollable tremor, postural imbalance, rigidity and slowness of movement3. The severity of motor symptoms is due to the
loss of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the SN4. Presently, PD therapeutic options available only
address symptoms and primarily include dopamine replacement therapy with levodopa (L-DOPA)5–9. L-DOPA was approved for
the treatment of PD in the late 1960s and is the most widely used prescription drug for PD10,11. Since L-DOPA/carbidopa therapy
only helps to control motor symptoms of PD and does not prevent or slow disorder progression, it is important to advance understanding
of idiopathic PD onset and progression.

 

The
specific etiology of PD remains unknown although there are genetic and environmental factors linked to disease development. Several causative
monogenetic mutations have been identified12–14. However, only a small percentage of cases (~10%) are the result of
inheritable genetic mutations1. The remaining cases (~90%) are sporadic or idiopathic in origin, although evidence suggests
that exogenous toxins such as cyanide, trace metals, organic solvents and exposure to pesticides increase the risk of developing PD15–19.
Epidemiological studies have demonstrated a strong link between environmental pesticide exposure with increased incidence of PD in humans20–26.
Indeed, idiopathic PD and the underlying epigenetic changes, have long been modeled using neurotoxins, including 6- hydroxydopamine (6-OHDA)
and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which all target dopaminergic neurons and lead to a parkinsonian syndrome.

 

Several
animal models exist that produce irreversible behavioral and molecular effects, such as those observed with this disease. The most extensively
studied animal model of PD uses 6-hydroxydopamine (6-OHDA). 6-OHDA is a hydroxylated analogue of the naturally occurring neurotransmitter,
dopamine, with a high affinity for the dopamine transporter and was the first model used to study PD 27. 6-OHDA is unable
to cross the blood-brain barrier so local injection is required in order to obtain the desired effects28,29. When 6-OHDA is
injected into the substantia nigra (SN) or medial forebrain bundle (MFB), it selectively accumulates in dopamine neurons resulting in
anterograde degeneration and cell death30. When 6-OHDA is infused into the striatum retrograde degeneration of the nigrostriatal
pathway also occurs causing a substantial loss of striatal dopamine and SN dopaminergic neurons31,32. Unilateral infusions
with 6-OHDA also produce considerable motor deficits as a result of dopamine depletion, the extent of which can be assessed by evaluating
rotational behavior following administration of either amphetamine or apomorphine28,33. Unfortunately, 6-OHDA does not induce
all pathological features of  PD, as  α-synucleinopathy and the formation
of Lewy bodies do not occur27,34.

 

The
link between mitochondria and Parkinson’s disease was first postulated in 1979 after a college student had attempted to synthesize
the heroin analog 1-methyl-4-phenyl-4-propionoxypiperidine (MPPP), but accidentally contaminated the drug with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP), such that when he self-administered the drug, he began to suffer from Parkinsonism-like symptoms 25. This effect was
observed again in 1982 when a young group of illicit drug users developed very progressive parkinsonian symptoms following intravenous
administration of MPPP that was contaminated with MPTP 1,35. Research revealed that MPTP can easily cross the blood-brain
barrier where it is oxidized into 1-methyl-4-phenylpyridinium (MPP+) by the enzyme monoamine oxidase B 36. Due to its similarity
in structure to dopamine, MPP+ is taken up by the dopamine transporter, leading to inhibition of complex I (nicotinamide adenine dinucleotide
(NADH)-ubiquinone oxireductase) activity of the electron transport chain and, ultimately, cell death 30,37. Furthermore, animal
studies revealed that

 

    	Confidential	4

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

MPTP
and other inhibitors of mitochondrial function, such as the pesticide rotenone, produce many of the same characteristics observed in
Parkinson’s disease including behavioral deficits, nigrostriatal degeneration, and protein aggregation 38–45.

 

MPTP
can cross the blood-brain barrier due to its high lipophilicity, where it is oxidized in glial cells and serotonergic neurons to 1-methyl-4-phenyl-2,3-dihydropyridinium
(MPDP+), catalyzed by monoamine oxidase B 46. MPDP+ is very unstable and spontaneously oxidizes to MPP+. MPP+ is then released
into the extracellular space and taken up via the dopamine transporter, where it targets mitochondria and inhibits complex I activity
of the electron transport chain. This leads to a decrease in the production of adenosine triphosphate (ATP), while increasing the generation
of reactive oxygen species, resulting in neuronal cell death 27,30. MPTP is one of the most commonly used animal models for
this disorder, as administration of this toxin results in motor symptoms and dopaminergic deterioration along the nigrostriatal pathway
that are very similar to that observed in Parkinson’s disease 47. The shortcomings associated with this technique include
an inconsistency in the loss of other monoaminergic neurons, such as those located in the locus coeruleus, and a lack of Lewy body formation
34.

 

Due
to the current level of understanding of the disease, diagnosis of patients with PD only occurs once individuals exhibit dysfunctional
motor symptoms, indicating that those suffering from the disorder have already lost at least 50% of their dopaminergic neuron population
in the SN and up to or exceeding 80% of the dopaminergic content in their striatum48. Therefore, preventative therapies or
strategies for preventing the onset or slowing the rate of progression are necessary for the treatment of PD, however such interventions
are not currently available. As mentioned previously, the therapeutic options currently available only address either motor or non-motor
symptom management, and primarily include the use of the hallmark dopamine replacement therapy, levodopa (L-DOPA). L-DOPA was approved
for the treatment of PD in the late 1960s and is the most widely used prescription drug for PD10,11. L-DOPA is absorbed by
the intestines, crosses the blood-brain barrier, where it is converted into dopamine in the brain. It is typically combined with carbidopa
or catechol-O-methyltransferase (COMT) inhibitors which prevent the breakdown of L-DOPA before it can reach the brain, prolong the duration
of action of L-DOPA and limits its side effects. Treatment with L-DOPA/carbidopa or COMT inhibitors can be effective throughout the course
of the disease, but due to the progressive nature of PD, increased dosages of the drug may be required over time to help control symptoms49,50.
Motor complications are very common with chronic L-DOPA treatment, with up to 50% of patients experiencing some form of motor dysfunction
within 2-5 years, and between 80-100% reporting symptoms after 10 years of therapy. These motor symptoms primarily take the form of the
development of dyskinesia (involuntary movements) and/or motor fluctuations, where symptoms are not controlled and can come on gradually
(L-DOPA effect wears off before the next dose) or suddenly and unpredictably51–53.

 

Beyond
motor symptom management with the highly regarded combination L-DOPA/carbidopa, PD patients may also be prescribed other common treatments
for motor impairments which include: dopamine agonists (mimic the effect of dopamine in the brain, can be used alone or in combination
with other PD medications, including L-DOPA, and are less likely to lead to negative motor side effects, as compared to L-DOPA, but are
also less effective for controlling motor deficits), monoamine oxidase B inhibitors (prevents the breakdown of dopamine in the brain
by reducing the activity of the enzyme monoamine oxidase, can be used alone or in combination with other treatments, and offer mild symptomatic
benefit in early cases of PD where the use of dopaminergic therapy is wished to be delayed or avoided), anticholinergic drugs (acetylcholine
levels are increased in PD and these treatments restore the balance between this neurotransmitter and dopamine, which act to help control
tremor and sometimes dystonia) and amantadine medications (act on the dopamine and glutamate neurotransmitter systems and are used to
treat dyskinesia and mild PD symptoms). Another treatment option available for some patients with poorly controlled motor fluctuations
is deep brain stimulation, where wires are implanted into targeted basal ganglia regions and stimulated using a remote device. The use
of deep brain stimulation has shown to be effective at reducing motor symptoms and improves overall quality of life, although there are
potential negative side effects associated with the procedure including speech and cognitive impairment, altered gait, and neuropsychiatric
sequelae, along with surgical risks54–60. In addition to the motor problems associated with PD, there are non-motor
symptoms which can be a result of the disease itself or due to the medications used to treat the disorder. Such non-motor complications
include constipation, dementia, depression or anxiety, drooling, fatigue, orthostatic hypotension, pain (directly relating to the disease,
muscle pain from dystonia or bradykinesia, or injury from falls as a result from balance or walking issues), psychosis (hallucinations
or delusions), sexual dysfunction, sleep disturbances, and urinary symptoms that can lead to incontinence58,61–63. This
current lack of available therapies for PD patients to slow or halt the rate of progression is disparaging and are necessary for the
treatment of PD, however such interventions are not currently available, thus urging the need for better therapeutic treatment options.

 

    	Confidential	5

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

Several
small molecule libraries were screened for protection in differentiated SH-SY5Y (dopaminergic neurons, verified using dopamine markers)
treated with 6-OHDA or MPTP. Several hit compounds were found and upon validation there were several outstanding hits, but most interesting
was that of resveratrol. Trans-resveratrol is a naturally occurring phytochemical found in red wine, blueberries, and many other
fruits and legumes. In studies exploring clinical efficacy, it has been demonstrated that resveratrol must be administered at high doses
due to its low bioavailability. In a randomized double-blind phase II clinical trial, the Alzheimer’s Disease (AD) Cooperative
Study group demonstrated that administration of up to 2 grams of resveratrol per day to AD patients resulted in stabilization of Aβ40 and Aβ42 accumulation in both CSF and plasma64. This
12-month trial began with a dosage of 500 mg daily, increasing by 500 mg every 3 months to a maximum of 1 g b.i.d. Initial dosages in
this study were likely inadequate, as positive effects were obtained after only 6-12 months of resveratrol treatment; population pharmacokinetics
(PK) evaluation shows that even at the highest administered dose at steady state, only about 200 ng/ml of plasma resveratrol were achieved
at Cmax. Literature reports have detailed the poor bioavailability of resveratrol due to first-pass metabolism65. To pursue
this research, the investigators determined that a significantly higher bioavailability or oral resveratrol would be required to safely
achieve therapeutic benefits. In the 12-month AD resveratrol study, side effects included weight loss and gastro-intestinal problems
such as nausea and diarrhea that are associated with high dosage. From a pharmacodynamic viewpoint, only high dose resveratrol has achieved
therapeutic benefit; importantly, some studies provide population PK data. In Friedreich’s Ataxia, a rare genetic disorder that
leads to progressive nervous system degeneration and motor deficits due to mitochondrial dysfunction, positive clinical (neurological)
outcomes were achieved with 5 grams (!) resveratrol/day66. Plasma Cmax levels under 200 ng/ml did not show benefits. Further,
5 grams of resveratrol in healthy individuals gives an average peak plasma Cmax of 539±384 ng/mL, which is approximately 2.4 μmol/L,
which is inadequate to elicit any beneficial biological effects as the activity of resveratrol at the biological level is observed in
vitro at concentrations of 10-100 μmol/L67–69. Importantly for our purposes, resveratrol protected dopaminergic
neuronal cell death in the presence of rotenone in the in vitro assay by 3 standard deviations as compared to vehicle.

 

Trans-piceatannol
is a derivative of resveratrol, both of which have been found to be beneficial in mucopolysaccharidosis I (MPSI), an autosomal recessive
genetic disorder in which the alpha-L-iduronidase (IDUA) enzyme is greatly depleted or absent from the affected individual resulting
in a buildup of the glycosaminoglycans (GAGs) heparan and dermatan sulfate GAGs. The buildup of these polysaccharide sugars in lysosomes
leads to cellular disruption, inflammation, cognitive decline and others. Eventually organ failure occurs, followed by death70.
Resveratrol and piceatannol have demonstrated therapeutic potential by upregulating IDUA enzyme activity, although piceatannol has approximately
10-fold lower potency than resveratrol. While not much is known about piceatannol in humans, resveratrol has been extensively studied
in humans with at least 49 clinical trials completed or ongoing in adults. No clinical trials of piceatannol have apparently been performed
to date. No clinical trials of resveratrol have apparently been performed in children.

 

    	Confidential	6

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

Resveratrol
appears to be very safe for administration to humans. Resveratrol is available over the counter from sources such as for example GNC.
Available formulations for resveratrol include 500 mg capsules, 15 mg/ml DMSO injectable solutions, oral solutions and sublingual capsules,
though some of these are perhaps not from trusted sources or have validated drug content. There is also apparently a transdermal patch,
likely from an untrusted source, for which it is unclear what the time-lag, steady-state plasma levels, maximal exposure and dermal permeability
to resveratrol are, and therefore the transdermal delivery is the least favorable for achieving high blood/brain concentrations of resveratrol.
Injectable forms also appear to be quite low in initial concentration. This route is also considered suboptimal for once daily dosing.
Sublingual administration clearly has benefits to circumventing first-pass liver metabolism, but it is unclear whether resveratrol passes
into the bloodstream quickly enough to achieve high AUC and Cmax. Anecdotal evidence also suggests that sublingual dosing requires holding
the drug under the tongue for 30 minutes or more. This could become problematic with twice or thrice daily dosing, whereupon compliance
is expected to be low. Oral administration is therefore considered to be the optimal route for resveratrol administration. However, there
are problems with first-pass liver metabolism reducing the overall drug residence time (AUC) and maximal concentration in the blood
stream (Cmax; both are measures of how well the body takes up a drug and keeps it around, in this case the higher these are, the better).
Since resveratrol demonstrates myriad of properties relevant to the treatment of PD and showed potential utility at high doses in preclinical
studies, the clear problem became how to deliver resveratrol in a highly bioavailable form that is well tolerated. We evaluated several
advanced drug delivery approaches to address this problem. However, transdermal or trans-buccal delivery is infeasible and the high blood
levels required coupled with rapid metabolism make infusion or injection routes impractical.

 

Resveratrol
is poorly soluble in water, and after absorption in the gut is rapidly degraded by first pass liver metabolism65. Micronizing
resveratrol prior to ingestion can improve bioavailability and adding components e.g., as emulsions or liposomes, may provide some benefits.
The physio-chemical properties of resveratrol suggest absorption and bioavailability would be improved if it could be presented as a
lipid like nutrient. Lipids pass into the lymph and avoid first pass liver metabolism. When resveratrol is dissolved in a lipophilic
phase and is stabilized in the form of droplets in an aqueous environment in emulsions, resveratrol can be retained in a phospholipid
layer in liposomes. This can increase the bioavailability as compared to the native form, but formulations of this type, such as liposomes,
are extremely unstable and are not resistant to the gastric milieu.

 

We
therefore developed a novel oral formulation of resveratrol (termed JOTROL), which shows significantly better PK properties than the
current best version of resveratrol (micronized). JOTROL is a stable micellar formulation for oral administration for use as a pharmaceutical
product in which the bioavailability of resveratrol is enhanced and avoids first pass metabolism (the formulation was granted European
patent protection EP3468535). JOTROL provides a unique solubilization product consisting of resveratrol, a mixture of polysorbate 80
and polysorbate 20 as well as at least one medium-chain triglyceride and tocopherol. The structure of JOTROL is micellar with a size
of approximately 30 nm, like the structure of the naturally formed physiological mixed micelles containing water-insoluble compounds
in its core, which is enclosed by ambiphilic molecules. The higher bioavailability, which is obtained by the product micellation, is
based on the independence of this transport system from the limiting parameters in the formation of the physiological mixed micelle.
It is important to appreciate the product micellar character as an “encapsulation and transport medium”. Resveratrol micellar
formulation increases peak blood plasma levels at least 10-fold more than non-formulated resveratrol. Our preliminary data show that
JOTROL is pharmacologically active in rodent brain. A pilot human exposure experiment showed similar results. Importantly, a reduction
of body burden is also expected to result in fewer side effects. Further, JOTROL has been approved for clinical trials for MPSI, with
a view toward using it in other neurological diseases such as Friedreich’s Ataxia and Alzheimer’s disease, demonstrating
its potential utility to progress into clinical phases for a spectrum of neurological disorders.

 

    	Confidential	7

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

EXPERIMENTAL
APPROACH

 

Goal:
Evaluate Resveratrol and JOTROL Activity in Parkinson’s Disease (PD) Toxin Models

 

Objective
1: Validate the protective capacity of JOTROL in a unilateral intracranial MPTP model using motor behavioral testing.

 

The
activity of unformulated resveratrol and JOTROL will be validated in an intracranial MPTP model of PD and sham-infused C57/BL6
mice, this approach has been directly modeled based upon work that is currently in phase II clinical trials for other drugs71–74.
In this milestone, we will work to validate the overall neuroprotective capacity of JOTROL on general health and motor behavior. Firstly,
animals will be microinfused with either vehicle (sham-infused controls) or MPTP using stereotaxic surgery to deliver these insults to
both the substantia nigra pars compacta (SN) and medial forebrain bundle (MFB) of the nigrostriatal pathway. Animals are expected to
recover within 24 hours and wound clips removed 7-10 days post-surgery. Following surgery, animals will undergo a functional observation
battery (FOB) which is a noninvasive systemic neurological examination to detect gross functional deficits following exposure to chemicals.
Observations of the animals will begin with their general behavior in their home cage, followed by measuring the animal’s general
locomotion in an open-field test. Behaviors will be ranked (present/absent, or on a scale) and include body posture, activity, coordination
of movement, respiration rate, lacrimation, grimace, righting response, arousal, ease of removal, handling reactivity, body weight, urination/defecation,
and presence of stereotypy or self-mutilation75. For the open-field test, animals will be placed in a PVC apparatus during
their dark phase, and will be allowed 5 minutes for exploration, and 5 minutes for testing. During testing, locomotion, occupancy in
the outer/inner areas and rearing will be measured. Weight measurements will continue daily to continue monitoring the health of the
animals (Fig. 1).

 

 

 

Once
the general health of the animals has been established (no endpoints reached) we will begin assessing the animals’ functional ability
in several different motor and cognitive behavioral tests including:

 

Open
Field. Spontaneous activities of the mice will be examined in the open field for 15 minutes (5 min of habituation and 10 min for
testing). Both horizontal (locomotion) and vertical activities (rearing) are monitored by an overhead high-definition video recorder
secured to the ceiling and behavior will be assessed using Ethovision software (unbiased)40,41,76. During testing, the total
locomotion, occupancy in the outer/inner areas, wall climbs, and rearing will be measured, with those animals infused with a toxin most
likely to display deficits in rearing, increased wall climbs and time in the outer area near the wall. Those treated with JOTROL are
expected to display activities like those infused with vehicle (sham), with such dysfunctional motor behaviors abolished.

 

    	Confidential	8

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

Grip
Strength. Animals are placed in the center of a wire mesh screen consisting of 12 mm squares of 1 mm diameter wire. The screen is
rotated to an inverted position 40 cm above a darkened padded surface (so the animal cannot see the ground). The amount of time from
the point the animal reaches the inverted position until the mouse falls off, or when the animal is removed because the criterion time
of 180 seconds has been reached, will be recorded and the average of three trials is used for analysis (Fig. 2)77. Animals
infused with a toxin are expected to demonstrate reduced grip strength. We anticipate that JOTROL will improve the grip strength deficit
that occurs in PD models.

 

 

 

Rotarod.
Mice are placed on the rods at 4 rpm and then accelerated over 30 seconds from 4 to 40 rpm. The maximum velocity (rpm) and the latency
to fall (min) is recorded. Animals are tested twice with approximately 30 minutes between each trial and the average is used (Fig. 3)78.
Animals infused with a toxin are predicted to display poor time and speed during testing, however successful treatment with JOTROL should
eradicate any motor concerns.

 

 

 

L-DOPA-responsive
Behavior. Each animal will be tested for response to L-DOPA by first injecting carbidopa (2 mg/kg, i.p.) 20 min before administering
L-DOPA (20 mg/kg, i.p.), 3 weeks after surgery then every 3 weeks throughout the duration of the study. Rotarod activity will be conducted
as described above, between 30 and 120 min after receiving the L-DOPA injection (well within the therapeutic window of L-DOPA88).
To assess locomotor activity and forelimb asymmetry, animals are placed in the open field apparatus for 10 min to reach baseline activity
levels. Animals will then receive carbidopa (2 mg/kg, i.p.) 20 min before administering L-DOPA (20 mg/kg, i.p.) and then returned to
the same apparatus for a further 120 min. L-DOPA is expected to temporarily restore motor function to near normal levels in toxin-infused
mice, so no changes or any other improvements are expected with JOTROL treatment.

 

    	Confidential	9

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

Rotation-induced
Behavior. Two weeks following surgery, animals will be injected with methamphetamine (5 mg/kg, i.p.; we hold necessary DEA
licensure and currently have methamphetamine in house) and recorded in
the same PVC apparatus as the open-field test. Mice will habituate to their environment for 10 min before contralateral and ipsilateral
turns are recorded for 90 min (Fig. 4)29,82. This test will be conducted no more than bi-weekly to reduce desensitization
to the drug. We expect JOTROL treatment will reduce the number of rotations in response to methamphetamine (sham-infused animals are
not expected to rotate).

 

 

 

It
is important to note that all testing will be conducted during the wake cycle of mice (at night, during their dark phase) and experimenters
will be blinded to treatment conditions.

 

High
plasma concentrations may be required and therefore we will work to secure large quantities of JOTROL. This may therefore necessitate
having it synthesized by a chemistry lab and if so then, overall quality must be high with a purity expected to be >99.5%.

 

Readouts
from daily JOTROL treatment are expected to have a positive impact on behavioral assays by attenuating motor deficits and neurological
dysfunction resulting from the unilateral infusion of MPTP. Resultant behavioral effects will provide fundamental insight into the underlying
mechanisms that are involved in governing the key mechanisms involved in PD pathophysiology and provide crucial knowledge about which
neuromolecular pathways are relevant.

 

Objective
2: Validate protective capacity of JOTROL on dopaminergic neurons in the SN and dopamine content in the striatum.

 

 

The
neuroprotective power of JOTROL will be determined by assessingany altered circuitry patterns in the unilateral MPTP model of PD
and control animals that were treated with vehicle or JOTROL. In this sub-milestone, animals will be evaluated
for dopaminergic integrity along the nigrostriatal pathway and dopamine content in the striatum. This will be conducted as follows:

 

Protein
Extraction and Western Blot Analysis of Dopaminergic and GABAergic Markers and Cell Populations in ex vivo Brain Tissue. Animals
will be anesthetized and rapidly decapitated for rapid brain harvest. Brain dissections will be conducted on a sterile petri dish over
ice to retrieve separate hemispheres of the midbrain (SN) and striatal tissue. Samples retrieved will be stored at -80°C until testing.
Samples will be rinsed and lysed using Mammalian Protein Extraction Reagent (MPER) supplemented with Halt Protease Inhibitor cocktail
(Thermo Scientific). The samples will be frozen for at least 6 hours and thawed to promote lysis, and the samples will also be sonicated.
Samples will then be centrifuged for 10 minutes at 14,000 x g. The supernatant will be transferred to new tubes and Bicinchoninic Acid
(BCA) assay (Pierce) will be conducted and measured using the EnVision plate reader (PerkinElmer) to quantify protein.

 

    	Confidential	10

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

For
western blots, the Criterion Blotter Western Blot system will be used (Bio-Rad). 30 ug of total protein in Laemmli sample buffer
will be boiled for 10 minutes at 95-100°C. The samples will be loaded into 10% gels (Bio-Rad) with electrophoresis set to
60-100V. The protein in the gel will be transferred to PVDF membranes by wet-transfer at 100V for 30 minutes. The membranes will be
blocked in 5% milk for one hour at room temperature, probed with primary antibodies overnight at 4°C in 5% BSA and with
secondary antibodies for 1 hour at room temperature in 5% BSA. The blots will be visualized by C-DiGit Blot Scanner (Li-Cor) and the
images quantified using ImageJ. Primary antibodies for dopaminergic and GABAergic populations and markers will be used including
anti-TH, anti-DAT, anti-NeuN, anti-parvalbumin, anti-somatostatin, anti-dopamine receptor 1, anti-dopamine receptor 2, and β-actin
(control). Secondary antibodies for each primary will be used for visualizing these proteins (Fig. 6)40.

 

HPLC
Analysis. To assess dopamine and GABA content in the striatum, we will again assess the fresh tissue samples collected as listed
above. Striatal tissue will be lysed in 0.5 M perchloric acid. The levels of dopamine and GABA will be measured using the Reversed-phase
Ultimate 3000 HPLC system with ECD detector and a reversed-phase column and analysed under the control of ChromeleonTM 7.2 Chromatography
Data system. The mobile phase is a mixture of 1.3% NaAc, 0.5% sodium 1-heptanesulfonate, 0.01% EDTA (adjusted to pH 4.0 with 100% acetic
acid), 2% methanol (v/v), and 7% Acetonitrile (v/v). All solutions for HPLC analysis will be double filtered through a 0.2 μm membrane
and degassed before use, and the flow rate will be set to 1 mL/minute.

 

SUMMARY

 

We
present here an enabling plan for clinical trials to evaluate JOTROL for the treatment of Parkinson’s disease (PD). Resveratrol
and JOTROL appear to be safe, and any positive outcomes will serve to enable PD clinical trials. Milestones presented here are accompanied
by go-no-go criteria, and costs associated with additional milestones will be discontinued at any time should such criteria not be met.

 

    	Confidential	11

    	 

    

 

Center
for Therapeutic Innovation, University of Miami Miller School of Medicine

 

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EXHIBIT
B

Budget

 

    	 

    	 

    

 

BUDGET

 

	PERSONNEL	 	7/1/2022 - 6/30/2023	 	Effort	 	 	Total	 	 	Total	 	 	 	 
	NAME	 	ROLE IN PROJECT	 	%	 	 	Salary	 	 	FB	 	 	TOTALS	 
	Shaun Brothers	 	PD/PI	 	 	3	%	 	 	4,900	 	 	 	1,343	 	 	 	6,243	 
	Candace Carriere	 	Post Doc	 	 	20	%	 	 	12,645	 	 	 	3,465	 	 	 	16,110	 
	 	 	 	 	 	 	 	 	 	Total personnel	 	 	 	 	 	 	 	22,353	 
	EQUIPMENT	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 
	Digital mouse sterotaxic fame	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	8,495	 
	Equipment total	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	8,495	 
	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 
	SUPPLIES (Technical)	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	8,237	 
	Supplies subtotal	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	8,237	 
	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 
	OTHER EXPENSES	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 
	Gas anesthesia mask/earbars/infusion pump	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	3,365	 
	Animal related expenses	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	7,966	 
	Other Expenses subtotal	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	11,331	 
	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 
	TOTAL DIRECT COSTS	 	 	$	50,416	 
	 	 	 	 	 	 
	TOTAL INDIRECT COSTS (53.5%)	 	 	$	22,428	 
	 	 	 	 	 	 
	GRAND TOTAL	 	 	$	72,844	 

 

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