الفهرس 
Title pageOnly 14 pages are availabe for public view
 |  | from | 125 |  |  |
| | | from | 125 | | |
Abstract
Toll Like Receptors (TLRs) are considered by some scientists as a ‘Swiss Army’
knife of the immune system, ready to respond in numerous states of infection and disease,
as in the last two decades’ cumulative evidences have linked TLR activation with the
pathogenesis of immune diseases and cancer. It has been brought to attention the
importance of the use these aspects of TLR signaling in therapeutic modalities to
accelerate and enhance the induction of vaccine-specific responses and target TLRs with
the use of biologics and small molecule inhibitors for the treatment of disease.
Furthermore, following the declaration of the World Health Organization (WHO)
of the H1N1 (swine flu) as the first influenza pandemic of the 21st century on June 11,
2009, the development of a vaccine to combat this pandemic relied mostly on the
understanding of the critical role of TLR in development of protective immune response,
which resulted in a vaccine developed by VaxInnate corporation and based on a TLR
mediated immune enhancement mechanism, as it contains sequences from the TLR5
agonist flagellin which interact with host TLRs to enhance immunological potency.
Specific TLR agonists have to be designed with reduced toxicity but increased
potency, as compared to traditional adjuvant candidates in order to fulfill the stringent
safety criteria required for prophylactic vaccines. The principal cellular targets of these
vaccines include APCs, primarily the DCs which link initial innate responses with
subsequent adaptive immune responses. TLR agonists designed for vaccines against
cancer and chronic viral diseases function by enhancing CD8+ T cell responses to protein
antigens, by cross-presentation of peptides generated from exogenous antigens or by
overcoming self-tolerance.
This study aimed at preparing three series of functional conjugates of the TLR7
agonist 420 attached through linkers to polycations, and to evaluate the ones with the
most in vitro TLR7 agonist activity for anti-cancer activity by intratumoral injection in a
murine solid tumor system.
In this study, series of TLR7/8 agonist compounds were prepared through
chemical synthesis of analogs to the lead compounds resiquimod (4-Amino-1-isobutyl-1H-imidazo[4,5-c]quinoline) and imiquimod (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine). Each compound was tested for its agonistic activity, anti-tumor effect, and
subjected to polycation conjugation trial for enhancement of its proposed agonistic
activity.
17 compounds were tested including resiquimod and imiquimod with molecular
weight ranged from 240.3 g/mol at its least value to 470.6 g/mol at its highest, with a
mean of 383.85±13.61 g/mol.
In order to assess the TLR7/8 agonistic activity of the compounds, the tested
compounds were assessed for the proposed agonistic activity in comparison to the known
dual TLR7/8 agonist; resiquimod, and single TLR7 agonist; imiquimod.
The optical density (OD) readings obtained by spectrophotometer at 630 nm were
analyzed using GraphPad Prism™ program to calculate half maximal effective
concentration (EC50). The EC50 values were used as a measure of potency of each
compound tested and were compared to the EC50 of both resiquimod and imiquimod
when tested on designed cell lines; HEK-Blue™ hTLR7, HEK-Blue™ hTLR8, and
HEK-Blue™ mTLR7, for studying the stimulation of TLR7/8 by monitoring the
activation of NF-κB and AP-1.
The compound 420 has the most potent agonistic activity in comparison to
resiquimod with a p value of <0.001 using a two-way ANOVA test with Dunnett’s
multiple comparisons test to compare each compound with resiquimod.
In order to assess the TLR7/8 agonistic activity of the TLR7/8 agonist -linker-polycation conjugates, the conjugates were assessed for the proposed agonistic activity
in comparison to the known dual TLR7/8 agonist; resiquimod, and single TLR7 agonist;
imiquimod.
The optical density (OD) readings obtained by spectrophotometer at 630 nm were
analyzed using GraphPad Prism™ program to calculate half maximal effective
concentration (EC50). The EC50 values were used as a measure of potency of each
conjugate tested and were compared to the EC50 of both resiquimod and imiquimod when
tested on designed cell lines; HEK-Blue™ hTLR7, HEK-Blue™ hTLR8, and HEK-Blue™ mTLR7, for studying the stimulation of TLR7/8 by monitoring the activation of
NF-κB and AP-1.
The polycation conjugates showed a comparable agonistic activity in comparison
to TLR7/8 agonists with respect to EC50 values retrieved. The comparison didn’t show a
significant difference using a two-way ANOVA test with Dunnett’s multiple comparisons
test to compare each conjugate with its respective TLR7/8 agonist.
In order to assess the anti-proliferative effect of the TLR7/8 agonists and CNT,
the assay was done on a flat-bottom 96-well plate and each compound was done in
triplicates; assigned to three wells. In respect to carbon nanotubes, the assay was done
for the HEK-293, hTLR7, hTLR8 and mTLR7 cell lines.
The TLR7/8 agonists were dissolved in 100% ethanol to reach the desired starting
stock concentration. The optical density (OD) readings obtained by spectrophotometer
at 540 nm were analyzed using GraphPad Prism™ program to calculate the 50% lethal
dose (LD50) or the 50% cytotoxic dose (CD50); the effective concentration of the
compound which induces a 50% reduction in cell number relative to the no-sample
control.
The anti-proliferative effect assay of TLR7/8 agonists showed a no activity
pattern of resiquimod in contrast to both imiquimod and compound 420 which show anti-proliferative activity, which adds a potential for the usage of compound 420 as anti-cancerous therapeutic agent.
On this ground, we can indicate the TLR7/8 agonists design is a necessity for
developing a new therapeutic model and the conjugation of such agonists is a tool for
desired effect augmentation in treatment of solid tumors as indicated by the anti-proliferative effect exerted on different cell lines.