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T

DEVELOPMENT OF AN ORAL FORMULATION OF SCV-07 FOR USE IN TUBERCULOSIS

ISTC Project No 2788

Participant: SciClone Pharmaceuticals CRADA # ORNL 03-0676

ABSTRACT

An evaluation of the immunomodulatory peptide SCV-07 was conducted as a

possible therapeutic treamenl for tuberculosis. Tlus evalualion included mouse models,

clinical t ia l s and various forms of the drvg such as liquid injection and development of

an oral pill. It was found h a t SCV-07 sigruficanlly increased the survival rale of animals

infected with lelhal doses ofMycobocterium bovis. It enhanced the functional activity of

macrophages in a dose-dependant fashion. The combhalion of SCV-07 with

bacteriostatic drugs, such as izoniazjd, was padlicularly effective. Phase J I clinjcal kids

in a TB clinic demonstrated that the usage ofthe injeclion form of SCV-07 for lung TB

treatment in combination with standard chemotherapy decreased the quantity of patients

w i h positive sputum assays for Mycobacteria, promoted healing ofcavities in lungs,

stabilized parameters of cell immuniiy, and resulted jn a significant improvement i n h e

general condition of patients. Clinical trial results of the oral drug form are still being

eva t ua~ed .

OBJECTIVE

The general objective of h s research project is h e development and evatualion

of the therapeutic potential of oral drug fonns of ~e immunomodulalory peptide SCV-07

fP n

for the treatment of tuberculosis (TB) since h s fonn j s the safest and most convenient

drug formulation for clinical and ambulatory usage. Extended clinical trials o f the

injeclion form of SCV-07 for the treatment of non-pulmonary TB wcrc also conducted.

Tuberculosis (TB) is a chronic infectious disease caused by the tubercle bacillus

Mycobuctcriirrn - (including M. tuberculosis, M. b o w , M. ufricunum, and M. microti),

w h c h worldwide affiits approximately 30% of the world’s 5.7 billion people. More than

3 million people die of TB each year and TB kills more people today than does any other

infectious agent. Some health experts view today’s TB situation as similar to AIDS in the

1980 with TB quickly reachng pandemic proportions. This highlights why the

development of new diagnostic methods, improvements in vaccine quality and a search

for new therapeulic approaches for treatment of TB are af great importance.

Pulmonary TB i s transmitted by the respiratory route. The Ikelihood of infection

is determined by the severily of the infection in h e infected person, h e exlent of

exposure (duration, amount ofcoughing, degree of f i l tering or dilution of’air, etc.), and

h e immune status of the infected person. I f the infected person has a competent immune

system, the growth and spread of bacteria is brought under cont~ol w d u n two lo Len

weeks. However, even in these cases, viable bacilli remain in the macrophages in a

dormant state, and reactivation of the disease can occur at a later date.

Persons with weakened immune systems are particularly susceptible to TB, and

infection with the human immunodeficiency virus (HW) weakens the body’s immune

system, HrV- positive persons have a much hgher risk oTdevcloping active clinical TB.

TB and H I V are synergistic infections - HIV d e c t i o n speeds the progression of the TB

disease and increases the activation rate of latent TB infection. TB appears to increase

HIV replication jn HJV jnfEtsd indivjduals. The World Health Organization esiimates

tha t more than 5 million people world-wide are co-infeded with HIV and TB, and

approximately one-third of all HIV-related deaths world-wide are caused by TB.

Upon an Initial infedon by he TB microorganism, alveolar macrophages ingest !he

tuberculosis bacilli and transport them to regional lymph nodes. T lymphocytes become

activated and release cytokines, interleuluns and chemotactic factors. Peripheral

monocytes enter the area and are transformed inlo aclivated macrophages by rhese

secreted faclors. Finally, the lymphocytes and macrophages wall off the site of infection

to form a granuloma. Ths process of cell-mediated immunib requires approximately 3-9

weeks. Due to the eficacy of h e process about 90% of in fe t ed people never become

clinically i l l . However, defects in immune funclion greally increase the chance that

clinical infection will occur. Moreover, clinical disease may recur i f there is a reduction

in pr)rmal immune funclion.

The response of the h u m a n immune system to antigens, including infectious agents.

is directed by two di ffermt subsets of T-helpcr cells (referred lo as type 1 and type 2, or

Th 1 and Th2). The predominance of either the Th I or Th2 lymphocyre subset can result

in different inflammalory pathways and disease outcomes. Research has shown [ h a t in

mycobacterial infections, i t i s the CD4+ Th 1 cells and their associated type 1 c y t o h e s

(interleukm 2 [lL-2] and interferon y [IFNy]) which play a key role boih in protective

m u m t y and immunopahology. IFNy is considered to be the major macrophage

activating factor, w h c h also plays a major role in suppressing a potentially disease-

promoting Th2 immune response.

n n

Multiple chemotherapeutic drugs for treabnent af TB are available, including

isoniazid, rifampin, pyrazinamide, and ethambutol. There are major drawbacks to these

therapies however, including the prolonged length of treatment (>6 months) and a

number of serious adverse reactions (including hepatitis, drug interactions,

numbnesdtingling/pain in extremities, fatigue, flu-]&e symptoms, bleeding, rashes,

stomach upset, and visual problems). In addition, and most importanlly, the emergence

of strains whch are resistan! to these agents have lead io dramatic outbreaks of

multidrug-resistant disease (MDR-TB) - most notable in the HIV-infected populaiions in

both the US and Europe.

SCV-07 (gamma-glutamyl-tryptophan) is one of a group of new

immunomodulatory compounds that possess gamma-glutamyl or beta-aspartyl moieties,

which were developed and patented both for composition and imrnunomodulatory use in

Russia (palent Nos. 2091 389 and 2 120298 [ 1 1 /28/95)]) and the USA (paten1 Nos.

5,744,452 [4/28/98] and 5,916,378 [ 1/29/99]). As Ihe most potent analog, SCV-07 has

been shown IO have a broad spectrum of immunostimulatory acljvilies both it, vitro and

in vivo tha t can affect the differentiation of pluripotenl stem cells lo thymocytes andlor

activation of Ihymocytes into T-cells with preferential aclivaljon of the Th1 lymphocyte

subset. SCV-07 has been shown to increase Can-A-inducd proliferation of thymocytes

and lymphocytas, stimulate expression of Thy-I, 2 antigen on bone-marrow cells, and

increase both production of It-2 and expression of IL-2 receptors in response to Con-A

in spleen lymphocytes. SCV-07 has also been shown to enhance the functional activity

of peripheral blood leukocytes. A strong immunostimulatory effect of SCV-07 has been

demonstrated in animals immunosuppressed by injection of 5-FU and in immunizatjon

n n

models with sheep red blood cells. SCV-07 injections in mice immunized with protein

antigen caused increased production of Th 1 cytokmes such as IL-2 and IFNy, but not of

T helper 2 cytakines. Ths implies that SCV-07 stimulates the immune response through

preferential activation of the Thl lymphocyte subset.

SCV-07 is thus appropnate as a therapeutic agent for infectious diseases

associated with an inadequacy of Thl-mediared Lmmunily, such as TB. SCV-07 is

particularly welt-suited for treatment of drug-resistant TB.

The primaq focus of the project was the development of an 9 3 - 0 7 drug

formulation for oral delivery. Th~s required: I) the development of immunological

models in experimental animals for evaluation of SCV-07 activity after oral

administration; 2) the development of an oral formulation of SCV-07; 3) an investigative

study af SCV-07 anti-tuberculosis activity after oral administration in combination with

tuberculostatic compounds; 4) evaluation of packaging and storage requirements; and 5 )

an evaluation o f disease progression in laboratory models of lung tuberculosis.

The methods used in h s project included classical immunology and bacteriology

t e c h q u e s as well BS molecular approaches:

- Lymphocyte er vivo proliferation studies using 3H-thymidinc technique - Lymphocyte surface molecule expression studies - Cytokine production in vivo and in cell cuhures using ELISAs and biological

- Th 1 and Th2 lymphocyte subset analysis using c y t o h e production patterns and

- PCR methods for delection of Mycobuclerizim ticbernilosis - A model of tuberculosis in mice, challengd with Mycoboclericint bovis

testing on cytolune-dependent cell lines

lg subclasses titers

Included in the project are expanded clinical rials of the SCV-07 injection

formulation in a TB clinic where the influence of SCV-07 on the clinical and

immunological status of patients suffering from lung tuberculosis was evaluated (as well

as the effect OP extra-pulmonary localization). The optimal schedule of SCV-07

administration was also determjned.

7 days post-irradiation WOC WIC RBC HCT

Sham M .036 ,120 7.72 38.1

Sham F .032 . I83 7.66 36.9

SCV-07 M .03 1 .I65 7.5 37.3

SCV-07 F ,093 ,142 8.34 41.1

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Additionally, a side experiment was also conducted to determhe the effectiveness

of 9 3 - 0 7 as a radioprotectant, since preliminary evidence indicated that stimulation of

h e h u n e system could afford some radiological protection.

Male and female C57BL61J mice were used for this study. For male and female

sham and male SCV-07, n=20; n=l5 for female SCV-07. Mce were injected

intraperitoneally (1 mgkg; 0.1 OSug/ul) on the day of irradiation (7Gy; Gobo source) and

every 3 days following irradiation. Sham mice received vehicle (PBS) alone. At 7 and

14-days after irradiation, 2 mice horn each group were bled for hematology analysis.

Mice were checked daily for deaths. At 17 days post-exposure, only a total o f 4 SCV-07

micc survived. A1 this time point, all remaining mice were sacrificed and blood counts

were analyzed.

WOC and WIC = 2 different means of measuring WBC counts RBC = red blood cell number HCT = hematocrit

m

A1 sacrifice (17 days post-irradiation)

Sham M (n=6)

n

WOC WIC RBC HCT

,045 A77 2.04 8.98

14 days post-irradiation I WOC

618

61 9

TOTAL

Sham M 1.037

1 1 3 5 I 2

14/20 14/15 17/20 14/20

Sham F 1.032

I SCV-07 M 1.024 SCV-07 M 1.024

I SCV-07 F 1.026 SCV-07 F 1.026

WIC WIC

,132

.08 1

,132

.08 1

,082 ,082 ,096 ,096

RBC IHCT I

3.59 3.59

Sham F (n-6) I ,082 I ,242 12-59 I 11.53

SCV-07 M (n=3) I ,141 I ,347 12.88 I 13.1 I SCV-07 F (n=1) 1.029 I nd I 1.44 16.04 I

The injection form of SCV-07 has passed a11 pre-clinical t r ia ls in Russia, and

phase I and J I c l in ica l b i d s have been successively completed. A l l required

documentalion has been submitred to the Pharmacological and Pharmacopocian

Committees. Permission for clinical usage of SCV-07 as a medicine for treatment of

secondary immunodeficiencies of different etiology was also granted. Additionally, SCV-

07 has bcea patented for bolh cornpsition and irnrnnnomodulatory use in bofh Russia

and the US.

Phase II clinical trials i n the TI3 clinic demonstrated that usage of the injection

fbrm of SCV-07 for lung TB beatment, in cornbination with standard chemotherapy,

decreased the number of. patients with positive sputum assays for Mycobacterium,

accelerated cavity healing, stabilized parameters oEcell immunity, and resulted in

s i p f i c a n t improvement of the patients’ general condition.

Success in the tasks outlined above led to a restricted clinical trial on volunteers

suffering from lung tuberculosis which is currently being finalized. During h s trial, in

which patients will receive SCV-07 in combination with anti-TB chemotherapy, both

clinical improvement and changes in lung condition are being evaluatd, a s well as the

immunological status of the patients.

The project is in conformity with the goals and tasks of ISTC and is aimed at

development of an immuostimulatory drug for therapy of infectious diseases and

secondary immunodeficiency. The results obtained from this project have extended

cooperation between researchers of Russia and the USA, and is contributing significantly

to the global prevention ofthe spread of tuberculosis.

Conclusions:

Investigation of the specific biological activity of SCV-07 in the experimental model of lung TB in mice demonstrated the ability of SCV-07 lo increase significantly the survival rate of animals infected with lethal doses of Mycobactevirrrn bovis. Tt enhar tcd the funclional activity of macrophages in a dose-dependant fashion. The combination of SCV-07 with bacteriostatic drugs, such as izoniazid, was particularly effective. Phase I 1 clinical trials in a TI3 clinic

demonstrated that the usage of the injection Tom of SCV-07 for lung TB treatment in combination with standard chemotherapy dccreasd the quantity of patients with positive sputum assays for Mycobacteria, promoted healing of cavities in lungs, s tabi l izd parameters of cell immunily, and resulted in a significant improvement in the general condilion of palienis. Addlion of SCV-07 to standard schemes o f T B therapy also helped io decrease the period of usage of hghly toxic chemotherapy drugs. Results (clhicd improvement and changes in lung condition as assessed by chest X-ray and bacteriologxsl studies) Gom usage oFthhc oral form of SCV-07 are still under investigation. The k~l low up period will be at least one year. No radioproteciant attributes were seen with SCV-07.