1 EXHIBIT 1
Investigaciones Biologicas y Quimicas
Dr. Juan Carlos Flichman
MEDALLA DE ORO UBA
MIEMBRO DE LA JUNTA CONSULTIVA DEL CENTRO ESPECIALISTAS
ANALISIS BIOLOGICOS DE BUENOS AIRES (CEABI)
CONTRA ENFERMEDADES TRANSMISIBLES SEXUALMENTE Y SIDA
ASESORA DE LA ORGANIZACION PANAMERICANA DE LA SALUD (OPS)
CONSULTOR HONORARIO PROGRAMA ETS DIVISION DERMATOLOGIA HOSPITAL DE
CLINICAS UBA
ReticuloseTM IN TREATING HUMANPAPILLOMA VIRUS (HPV)
OPEN LABELED, NON-COMPARATIVE, MULTI-CENTERED CLINICAL TRIAL TO
DETERMINE THE SAFETY AND EFFICACY OF RETICULOSE IN TREATING (HPV)
FINAL REPORT
ADVR-AR-96001
AUTHORS: FLICHMAN J.C.; BLUMTRITT C.; CASCO R.H.; TAUSCHER P.;
SPROVIERI O.; SOMMA E.; PIRES TORRES C.; LOWENSTEIN M.; VILLAREAL C.;
LEMA B; MACIEL A.
COMPLETE ADDRESS/INSTITUTION: U.A.C.E.T.S./HOSPITAL DE CLINICAS
UNIVERSITY OF BUENOS AIRES/HOSPITAL MUNICIPAL B. RIVADAVIA.
CALLE PASTEUR #740 CAPITAL FEDERAL (CP 1028) BUENOS AIRES, ARGENTINA.
TEL./FAX: (54-1) 951-2448
SUMMARY:
Twenty (20) persons (6 males and 14 females) were diagnosed to be
infected with HPVs. The presumed clinical diagnosis and infection was
confirmed by means of Papanicolaou, colpo-penis and/or anoscopy,
biopsy, and PCR. The patients were treated topically with
ReticuloseTM a solution composed of peptide nucleic acid (PNAs).
Based on the remissions and the markedly clinical improvement
observed in 50% of the patients, we can conclude that the PNAs in
ReticuloseTM may, as described by Hirschman S.Z. and Chen W., favor
the synthesis of gamma interferon, interleukin 1, interleukin 6, and
tumor necrotic factor alpha (TNF-alpha).
We recommend and encourage using ReticuloseTM as an alternative in
biological treatment in HPVs. It is non-toxic and considered to have
possible antisense activity or is an immunomodulator.
KEYWORDS:
Peptide Nucleic Acid, (PNAs), Papilloma Virus (HPVs), Condylomas,
SIL, Cervical Cancer.
INTRODUCTION:
The Genital infection, Humanpapilloma Virus (HPVs) is a Sexually
Transmitted Disease (STD) directly related to the number of sexual
partners, and exists in both sexes with latent and acute infection.
The number of consultations in the STD Centers can be five times
greater when compared to the Centers used by the general population
(1,2).
The infection is increased through homosexual transmission (3).
When evaluated through biochemical parameters (4), no doubts exist as
to the relationship between the evolution of HPVs infections and the
immune function, or that CIN (Cervical Intra-epithelial Neoplasia) or
SIL (Squamous Intra-epithelial Lesions) are strongly associated to
HPVs infections.
The HPVs have developed new strategies for their propagation and
survival, changing form asymptomatic to symptomatic with inflammatory
disorders and low or high grade SIL, the latter relating to cervical
cancer (5).
Investigations to determine the genotype is recommended to establish
the oncogenic relationship, that are the basic mechanisms that
involve this process, in order to control the HPVs infection and
treat the infected population (6,7).
METHODS & MATERIALS
An open label, non-comparative, multi-centered clinical trial was
conducted to determine the safety and efficacy of ReticuloseTM in the
local treatment of virus condylomas in the skin gland, anal, and
cervical mucous.
ReticuloseTM is manufactured by Advanced Viral Research, Corp., USA.
ReticuloseTM is a peptide nucleic acid solution developed by a
chemical process. Peptide nucleic acids (PNAs) are newly appreciated
molecules that have been shown to have interesting properties; for
example, they are very stable and have antisense activity (8).
Twenty persons, 6 males and 14 females of Caucasian origin, between
18 and 39 years of age, 3 married couples, heterosexual activity, non
reactive to HIV, CD (+) count above 350/mm3, low grade SILL and free
of flora anaerobic (Fa), Candidacies (Cand), Chlamidydia trachomatis
(CHT), and/or Mycoplasmas (MY), [M. Hominis, M. Fermentans,
Ureaplasma urealyticum], CHT was directly controlled (elemental
bodies) by monoclonal antibodies, and the remaining microorganisms
through specific cultures.
When these infections existed prior treatment was given: FA with
metronidazole 7 days/1 gram daily, Cand with fluconazol 150 mg
daily/2 days, GHT and/or MY with azitromicin 1 gram daily/7 days.
The patients were clinically diagnosed via: Papanicolaou, colpo -
pene and/or anascopy using 5% glacial acetic acid, biopsy and PCR
with primers, as per international consensus GP5/GP6 and TSMIX for
genotypes 6, 11, 16, 18, 31, and 33. The last four with a greater
oncogenic potential for cervical cancer.
ReticuloseTM was applied to 6 male patients topical in full strength
directly on the exphitic condylomas once a day/14 days. Fourteen
(14) females also received full strength topic application during
their scheduled medical visits, 2 per week/2 weeks for a total of 4
applications. In addition, the female patients were instructed to
peroform daily applications (except for when medical visits were
performed) using a vaginal applicator prepared with 10% of
ReticuloseTM in 6 grams of a hydrosoluble carbolic base.
Biochemical controls were performed pre and post treatment. In
addition the following was done: cervical vaginal swabs, routine lab
work: sediment rete, blood glycol, creatinine, hemogram with CBC
platelet count and urine analysis. Hepatic functions: total and
direct bilirubin, transminuses, and piruvic, lgG total, Protrombin
percentage, HbsAg, Anti Bc IgG, Anti HCV. Immunological profile:
CD4 (+), CDS (+) and Beta 2 microglobulin.
All exams were performed one week prior to treatment and one week
after completing the treatment. Total study time per patient
averaged 30 to 35 days.
RESULTS:
Table 1
Treatment
PRE POST
Vaginal Cervical Swabs
infected with
Fa/Cand Cht and/or My 5 0
Colposcopy 13 Acetowhite (+) 6 Acetowhite
(+)
Penescopy 6 Acetowhite (+) 6 Acetowhite
(+)
Anoscopy 2 Acetowhite (+) 2 Acetowhite
(+)
PAP Women 13 Class II II Class II
Histopathological Microscopy in Males
Coilocytea (exophitic condylomas) 6 6
SIL low grade 13 11
PCR genotype 11 9 8
PCR genotype 16 3 3
PCR genotype 18 3 3
PCR not identified 4 4
PCR negative 1 1
a) Diminished color intensity 4
b) Diminished size 2
c) Flattening 1
d) Softening 1
f) Clinical improvement: 8
g) Total remission: 2
h) Side effects 1
i) Reduced pain during sexual relations 14
In two of the 13 cervix studies had complete remission (1 PCR
negative from the start and 1 genotyped 11), the remainder continued
PCR positive conserving their genotype.
TABLE II
Please refer to this section in the report for detailed results per
patient.
DISCUSSION:
It was clinically determined that when ReticuloseTM was applied
topically (2/20) had total remissions, (8/20) experienced clinical
improvement ranging from: 4 reduction in color intensity, 2
reduction in size, 1 flattening, and 1 change in texture.
Until now, topical treatment in HPVs has consisted of trichloracetic
acid, podophyllin, podophyllin toxin, and interferon alpha, beta, and
gamma. Except for the interferons that have contraindications for
pregnant women, and in immunodepression, the active mechanisms used
in other treatments are not directed against HPVs, but rather against
the cells that contain it causing a reaction of necrosis, cellular
destruction, inflammation, irritation, and pain.
Interferons act as agents, and based on the remissions and clinical
improvements obtained via topic applications, we consider PNAs as
possible antisense agents, as indicated by Hirschman A.Z. and Chen W.
(9,10) where they found that PNAs stimulated production of chemokines
including IL1, IL6, gamma interferon, and TNF-alfa in cell cultures.
The efficacy of RETICULOSETM on HPVs may be due to its possible
antisense activity and that it is considered to be an
immunomodulator. Hirschman S.Z. (9) of Mt. Sinai Hospital in New
York, established that RETICULOSETM stimulates in cell culture
systems the production of a unique set of Chemokines, including
Interleukin 6 and Gamma Interferon. Gallo R. (10), demonstrated that
the cells in the immune system produce varied "hormone like" chemical
products, that block receptors to specific attacks on cells ("target"
cells).
In this aspect we can apply similar criteria to HPVs where in AIDS
new lymphocyte receptors CD4 ( ) were discovered in 1995 that permit
the entrance of HIV. The virus needs the receptors CCCKR5 and fusin.
If these receptors are bioblocked HIV is not permitted to enter. The
receptor fusin allows the fusion of CD4 ( ) and the entrance of a
more pathogenic phenotype: SHIV (opens the way for HIV to enter).
The biological blockers impede the virus from attaching and holding
on to the cell receptors acting as a drug that can limit the
propagation of infections specially in the early stages.
If the results obtain "In Vitro" are reproduced "In Vivo" we should
obtain increased results by administering RETICULOSETM via
subcutaneous or intramuscular injections.
Even if these Chemokines can not eradicate the infection, they may
impede the virus from mutating to a more pathogenic form. It may
occur on genotypes 16,18,31, and 33 when the antecedents indicated
low risk genotypes like 6 and 11.
No side effects were observed in any of the 20 patients while using
RETICULOSETM on skin or mucousal. Only one patient (with anal
condyloma) had an eritema that disappeared spontaneously within 24
hours. The patient had multiple allergy. The female patients
performed the self-application of the medication with total safety.
CONCLUSION:
1. In exophitic condylomas of the skin where the size is
appreciably large, and until we know the results of using the drug
intramuscularly, we recommend previous classic treatment with:
tricoloroacetic acid, podophyllin, podophyllin toxin, 5 flurouracil
(5FUO, cryosurgery, or laser as an initial phase. Continue with
RETICULOSETM within one week with topic applications directly on the
lesion every other day for a period of 30/60 days, 3 or 4 times a
year.
2. In cases where Condylomas are within the cervical mucousa
RETICULOSETM can be used without any previous classical treatment, as
the results have demonstrated the efficacy of RETICULOSETM in
treating HPVs.
3. As a result of the tolerance, increased quality of life, and in
particular the reduction of pain during sexual intercourse and the
ability to return to sexual relations at an earlier phase of the
treatment, we recommend the topic use of RETICULOSETM.
4. We find it is important to continue clinical trials using
RETICULOSETM via sublingual and/or subcutaneous/intramuscular
injection. These methods should increase the efficacy as it
is non-toxic, and considered to have possible antisense activity
and/or is an immunomodulator.
BIBLIOGRAPHY:
(1) Olmos L. Condilomas acuminados (verrugas genitales)II. Revista
Ibero-Latinoamericana de E.T.S., 4; no 3: 131 - 42; 1990.
(2) Mi o M., Hernandez Fl, Mu oz A.J., Torres L.M., Moreno L.,
Mendoza N.: Diagnostico de las infecciones por el virus del Papiloma
Humano. Revista Ibero-Latinoamericana de E.T.S., 9; no. 6; 393 - 6;
1995.
(3) XI International Conference on AIDS/Vancouver. Track B:
Clinical Science. Squamous Intraepithelial Lesions/HPV Infection in
men and women, July 10, 1996.
(4) Flichman J.C. EL SIDA, HPVs y Otras Enfermedades Transmisibles
Sexualmente. Ediciones Urano, 1988.
(5) Mauro Romero Leal Possos e Cols. Doencas Sexualmente
Transsiveis. Edit. Cultural Medica Ltda., 4ta. Edicao.
(6) Monsonego J., Dysplasies Du Col Uterio el Papillomavirus
Humains. Maloine SA. Editeur; 1988.
(7) Monsonego J., Papilloma virus in Human Pathology. Series
Challenges on Modern Medicine. Editor Eurogin; Vol. 9, 1995.
(8) Stephen C.B., Stephen A.T., James A.V., Donal G.D., NMR
Solution Structure of a Peptide Nucleic complexed with RNA. Science
265:777-80; 1994.
(9) Hirschman S.Z. Peptide Nucleic Acids Stimulate Gamma Interferon
and Inhibit the Replication of Human Immunodeficiency Virus. Journal
of Investigative Medicine, Am Fed Clin Res, com personal a publicarse
agosto 1996.
(10) Gallo R. XI International Conference on AIDS/Vancouver. Track
A: Basic Science. Chemokines, July 9, 1996.
Notas:
1. Zona de tranformacion de un Ectropion de la Mucosa
Endocervical (ZTEME) con infiltracion inflamatoria cronica del
corion, epitelio pavimentoso mataplasico, aglucogenico con
disqueratosis y vaculozacion perinuclear compatible con coilocitosis
sin displasia.
2. ZTEME con infiltracion inflamatoria cronica del corion,
epitelio pavimentoso metaplasico, aglucogenico con disqueratosis y
vaculozacion perinuclear compatible con coilocitosis sin displasia.
3. Zona de transformacion con epitelio escamoso metaplasico
con acontopapilomatois, aglucogenisis, hiperplasia focal de la capa
basal, vacuolizacion perinuclear compatible con coilocitosis sin
caracteres displasicos.
4. Cuadro histologico compatible con un condiloma plano
incipiente sin caracteristicas displasicas. No se observa
proliferacion atipica en el material estudiado.
5. ZTEME revestida por un epitelio pavimentoso maduro
anomalo, no displasico. Se observan celulas con discreta
vacuolizacion perinuclear pero sin caracteres morfologicos
especificos.
6. ZTEME revestido en parte por un epitelio anomalo no
displasico y en parte por un condiloma plano sin caracteres
displasicos.
7. Epitelio pavimentoso anomalo no displasico en sectores
compatible con un condiloma plano.
8. Zona de transformacion de un ectropion de la mucosa
endocervical, epitelio pavimentoso metaplasico compatible con un
condiloma plano.