Post Treatment Lyme Disease Syndrome Research
Scientific abstract:
Though the exact reason for the cause of PTLDS is not known but
there is unmet need for developing therapeutic agents. We have identified a
drug that kills both stationary phase and drug-tolerant Borellia burgdorferi (Lyme
bacteria) at 20 μg/ml. When tested in in vivo, the antibiotic has shown good
efficacy against B. burgdorferi infection in C3H/HeN mice model. Furthermore, the
antibiotic reduced macrophage infiltration to spleen after 7 days of B.
burgdorferi infection and decreases induction of M1 macrophages (NOS2+) with
IFN-G+ response, which helps for Lyme disease progression. In addition to it, the
antibiotic also reduced proinflammatory mediators responsible for Lyme disease
propagation. With a goal to treat Lyme affected patients with the antibiotic,
we have developed an oral formulation by encapsulating the antibiotic in
alginate beads using ionotropic-external gelation technique and then enteric
coated with eudragit S100. Our novel formulation has ability to protect the
antibiotic from acidic pH of stomach and releases drug only in intestine (at
basic pH). Based on our preliminary data, we propose we will study how the
immune system responds during infection and treatment with different drugs that
are currently used for treating Lyme disease and also comparing with our lead
molecule which has ability to kill persisters. We will also test our novel oral
formulation in rats and study pharmacokinetics (PK) and pharmacodynamics (PD).
A. Specific Aims
AIM 1:
Studying
immunomodulatory and anti-inflammatory mechanisms of the antibiotic in C3H
mouse model
Lyme disease is a multisystemic
infection which affects the heart, joints, skin, musculoskeletal and nervous
system. In humans Lyme disease develops inflammatory arthritis and carditis.
Immune cells like neutrophils and macrophages infiltrate into different organs
like heart, joints, lymph nodes and spleen etc, during infection and play a
vital role in disease progression and resolution. During Lyme infection,
macrophage phenotype is thought to play an important role with M1 macrophage responses
considered to be pro-inflammatory and M2 macrophage conditions are associated
with resolution of bacterial infection or persistence. Studying the role of
different types of macrophages will gives insights of disease development and
how these immune cells are modulated during treatment in different stages of
diseases. Innate immune cells after their exposure to B. burgdorferi induces
inflammatory mediators like cytokines and chemokines which are tightly
regulated by the anti-inflammatory cytokine IL-10. The cytokines and chemokines
regulate early innate immune responses, link innate and adaptive immunity, and
are thought to play key roles in the pathogenesis of Lyme disease. Studying
differences in their levels and patterns of up and down-regulation helps us to
understand survival of the spirochete during early infection and subsequent
disease progression.
To understand the role of
immune cells and inflammatory mediators (cytokines and chemokines), we study B.
burgdorferi infection at different time points like 7, 14, 21 and 56 days which
covers different stages (early, acute, late and chronic). Our specified study
is done how immune system responds during infection and treatment with
different drugs that are currently used for treating Lyme disease and also
comparing with our lead molecule which has ability to kill persisters.
AIM 2:
Identification of the antibiotic binding partners in B. burgdorferi.
To study the binding partner(s)
of the antibiotic, we will synthesize biotinylated derivatives for pull down
assays. We propose to treat bacteria l lysate with biotinylated antibiotic
derivatives. Post-treatment and cell lysis, the interacting partners of the
antibiotic from the cell hydrolysate will be isolated using streptavidin beads.
We will characterize the binding partners using mass spectrometry analysis. We
will also prepare the recombinant protein (binding partner) and carryout
biophysical studies to understand the nature of the interaction. Understanding
the interaction of the antibiotic and binding sites of B. burgdorferi related
proteins would greatly guide future iterations of antibiotic development for
more effective treatment of Lyme disease.
Aim 3:
Studying
pharmacokinetics (PK) and pharmacodynamics (PD) for oral formulations of the
antibiotic in rats for future use of clinical trials.
The antibiotic is one of the
semi synthetic penicillin classes of drug that have broad spectrum of activity
particularly against Pseudomonas aeruginosa. It is administered parentally by
slow intravenous injections or intravenous infusions. Oral delivery is
non-invasive and more amenable for patience compliance and reduced cost. Also the
antibiotic could not be effectively delivered orally due to its chemical
instability in low pH environments in the stomach reducing its bioavailability.
Our lead candidate is currently used for cystic fibrosis and is administered
via IV infusion. Alternate ways of delivering the antibiotic though lung has
being attempted with less effective outcome. So to overcome these problems, we
have developed an oral form of the antibiotic and tested it’s in vitro drug
release in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).
Based on these promising results of our oral formulation, we will study PK and
PD for oral formulation of antibiotic in Sprague Dawley rats for future use of
clinical trials
B. Significance and
Innovation
Lyme disease is caused by
Borrelia burgdorferi sensu lato which accounts for >90% of all vector-borne
disease cases in the United States and affects ~330,000 persons annually in
North America1. Traditional tetracycline antibiotic therapy is generally
prescribed for Lyme disease and is effective in treating disease2. However, 10
- 20% of patients treated with current antibiotic therapy still show lingering
symptoms of fatigue, pain, arthralgia, myalgia, and/or perceived cognitive
impairment. In some cases, these symptoms persist more than 6 months after completing
conventional Lyme disease treatment. This condition is referred as
Post-treatment Lyme Disease Syndrome (PTLDS)2,3. The existence of PTLDS is
debatable, some researchers consider the presence of persister forms of B.
burgdorferi and some argue it is due immunological response3,4,5. There is a
huge unmet need to treat PTLDS patients either clearing remaining persister
forms of B. burgdorferi or by alleviating immunological responses due to PTLDS.
To find an effective treatment we have identified the antibiotic from our high
throughput drug screening, which eliminates stationary phase B. burgdorferi
completely at 20μg/ml. In addition to it, the antibiotic also kills
drug-tolerant borrelia persisters effectively at 20 μg/mL and 40 μg/mL in in
vitro. The borrelia persisters generated due to its inability to eradicate by
doxycycline were also effectively killed by the antibiotic at 20 μg/mL and 40
μg/mL (personal communication). We have successfully shown that the antibiotic
effectively eliminated B. burgdorferi in in vivo C3H/HeN mouse model. In
addition to it, the antibiotic treatment reduced splenomegaly in mice infected
with B. burgdorferi for 7 days. The antibiotic also reduced macrophage
infiltration to spleen after 7 days of B. burgdorferi infection and decreases
induction of M1 macrophages (NOS2+) with IFN-G+ response, which are responsible
for Lyme disease progression. We have also observed the antibiotic treatment
reduces proinflammatory cytokines and chemokines like MIP-1b, IP-10, MCP-3,
TNF-A, IL-17A and IL-1A which are very essential to Lyme disease propagation.
From our preliminary results, we have observed the antibiotic in eliminating B.
burgdorferi completely and reducing proinflammatory mediators responsible for
Lyme disease progression.
In an attempt to take our
findings to the patients affected with Lyme disease, we have developed an oral
formulation for the antibiotic. This is mostly administered intravenously and the
antibiotic delivery by oral route is degraded by stomach acids at pH 7. In
order to protect the antibiotic in stomach at pH 2, we have encapsulated the
antibiotic in alginate beads using ionotropic-external gelation technique and
then enteric coated with eudragit S100. Our novel formulation protected the
antibiotic at acidic pH and released only at basic pH 7. From our studies we
were able to achieve the antibiotic is only released in SIF at pH 7 and
attained a concentration of 30 μg/ml in in vitro. Based on the evidences from
our studies, the antibiotic drug has huge potential to treat all patients
affected with acute Lyme disease and PTLDS. The antibiotic oral formulation
that was developed has ability to eradicate B. burgdorferi and can kill
borrelia more effectively than the currently prescribed drugs.
C. Approach:
Previous Work/Preliminary
Data
In our search to identify safe
and effective molecules to eliminate B. burgdorferi persisters, we have
screened 7450 chemical compounds (80% of them are FDA-approved) from several
different libraries, using a BacTiter-Glo™ Assay6. We focused on identifying
FDA-approved drug molecules so that the identified drugs can be repurposed for
the use of Lyme disease treatment effectively. To achieve this, we have
successfully screened several libraries and identified nearly 300 hit
molecules6,7. After studying the minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) of the 50 hits, the lead molecule was
chosen for further studies based on their ability to kill B. burgdorferi at low
concentrations and excellent safety profile.
The antibiotic, our top
candidate, successfully eliminated all B. burgdorferi stationary phase
persisters in an in vitro persister culture model6. In another experiment, we
performed a time-dependent in vitro study for the antibiotic along with mitomycin
C which is shown that it can eliminate persisters by sharma etal8. In control,
the Bb growth increased to 108 cells/ml. The antibiotic successfully eliminated
all B. burgdorferi like mitomycin C. From time kill studies, we observed that the
antibiotic completely eliminated B. burgdorferi in 96 hours.
In the current study, we
identified that the antibiotic completely kills B. burgdorferi taken from
stationary phase cultures. We have also observed that the antibiotic alone
kills doxycycline-tolerant borrelia persisters effectively at 20 μg/mL and 40
μg/mL (personal communication). Our in vitro results are also validated by
preliminary in vivo experiments, showing that the antibiotic and cefotaxime can
effectively eliminate an in vivo C3H/HeN mouse model9. Seven days post-infection
with B. burgdorferi, the mice were intraperitoneally administered a daily dose
of drugs, the antibiotic (50 mg/kg), cefotaxime (50 mg/kg), or ceftriaxone (50
mg/kg) for 5 consecutive days. After 48 hours of the last administered dose,
the mice were sacrificed, and their urinary bladders, ears, and hearts were
harvested and total DNA was isolated and Q-PCR was performed using Fla-b
(borrelia specific primers and probe) and by Delta CT method10. In all the mice
treated with the antibiotic and cefotaxime the B. burgdorferi infection was
completely eliminated. The antibiotic is well tolerated, up to the dose of 450
mg/kg/day11.
The antibiotic helps in
alleviating inflammation and Lyme disease progression
During Lyme infection
macrophages play a role in the development of both Lyme arthritis and carditis.
A significant proportion of the inflammatory infiltrate in infected joints and
hearts of B. burgdorferi infected mice is made up of macrophages. Macrophage
subsets mainly M1, M2 and rM (resolution macrophages) plays important role in
disease pathogenesis. To study immune regulation, experimental Lyme borreliosis
is an excellent model because mice develop an inflammatory arthritis and
carditis that peak and then resolve as the spirochetes are cleared from the
infected tissues. So far, not much studies were done to understand how
macrophages infiltration to heart, spleen and joints, during infection and
resolution of disease in the context of treatment with antibiotics. From our
studies by flow cytometry we have observed the antibiotic reduced splenomegaly
after 7 days of borrelia infection and followed by treatment with the
antibiotic for 5 days. The total no of splenocytes and F4/80+ CD45+ macrophages
were reduced when compared to borrelia infection and also with currently
prescribed antibiotic like doxycycline. We have also observed the antibiotic
reduced macrophages (Figure 3C) specifically M1 (F4/80+, NOS2+) with IFN-G+
significantly. As IFN-G+ promotes borrelia infection16, our preliminary data
showed the antibiotic helps in alleviating inflammation and Lyme disease
progression in mice infected for 7 days.
Like in other infections
cytokines and chemokines play key roles in Lyme disease pathogenesis. Upon
stimulation with B. burgdorferi, macrophages produce of several proinflammatory
cytokines, such as IL-12, IL-6, IL-1B, and TNF-A etc. Studying qualitative and
quantitative differences in inflammatory mediator’s production by macrophages
during infection and treatment with different drugs helps us to understand
their role in disease progression. In our study, we have observed treatment
with the antibiotic reduces proinflammatory cytokines and chemokines in mice
infected with B. burgdorferi for 7 days. The serum analyses by 38plex shows
several inflammatory mediators like MIP-1b, IP-10, MCP-3, TNF-A, IL-17A and
IL-1A were reduced significantly by the antibiotic. Based on our promising
preliminary results, we propose we would like to study the role of immune cells
in spleen, heart and joints. In addition to it, the role of cytokines and
chemokines in Lyme disease progression and resolution during treatment with the
drugs will be investigated.
In vitro targeted
release of the antibiotic oral formulation only in basic SIF at pH 7
This antibiotic is one of the
semi synthetic penicillin classes of drug that have broad spectrum of activity
particularly against Pseudomonas aeruginosa. It is administered parentally by
slow intravenous injections or intravenous infusions. Oral delivery is
non-invasive and more amenable for patience compliance. Also, the antibiotic
could not be effectively delivered orally due to its chemical instability in
low pH environments in the stomach reducing its bioavailability. We have
developed an oral form of the antibiotic and tested its in vitro drug release
in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). We have
encapsulated the antibiotic in alginate beads using ionotropic-external
gelation technique and then enteric coated with eudragit S100. The use of
sodium alginate, and eudragit S100 prevented the drug release behavior in
gastric conditions and helps in sustained release at intestinal pH 7. Our
formulation beads with enteric coating with eudragit S100, protected the
antibiotic from the harsh environment of the stomach. In our oral formulation studies,
we have observed that the antibiotic is not released in SGF at pH 2 until 120
min. The antibiotic is only released in SIF at pH 7 and attained a
concentration of 30 μg/ml. The concentration we have shown that the antibiotic
kills B. burgdorferi is only 20 μg/ml. It is very essential to study PK and PD
of these oral formulations in Sprague Dawley rats before planning Phase I
clinical trials. Our developed oral form of the antibiotic has ability to
eradicate B. burgdorferi and can kill borrelia more effectively than the
currently prescribed drugs.
Research Strategy and
Methodology
Strategy of immunomodulatory
and anti-inflammatory mechanisms of antibiotic study in in vivo C3H mice: Four
weeks old female C3H/HeN mice will be infected with 100,000 B. burgdorferi. On
the day 7, 14, 21 and 56 of infection, the mice will be administered a daily
dose of drugs, the antibiotic (50 mg/kg), cefotaxime (50 mg/kg) or ceftriaxone
(50 mg/kg) for 5 consecutive days by intraperitoneal route. After 48 hours of
the last dose of administering compounds, the mice will be sacrificed, and
their blood, spleen, heart, urinary bladders, ears and ankles will be
collected. Ears will be suspended in BSK-II medium to evaluate for the presence
of motile spirochetes after 21 days using the dark-field microscopy. We will
perform 38 plex assay and will analyze cytokine and chemokine from serum
collected. From ankle we will isolate bone marrow derived macrophages (BMDM)
and cultured with either 1mg/mL of LPS or with live B. burgdorferi at a
multiplicity of infection (MOI) of 10:1 for 24 h to determine cytokine and
chemokine responses with 38 plex assay. The cytokine and chemokine analysis at
different stages of Lyme infection will helps to understand responsible
molecules for disease progression and also to identify new makers for Lyme
disease. It is well studied that the IFN-G, IL-4 and IL-10 plays a major role
in modulation of inflammatory responses in Lyme disease9,14. So we will isolate
macrophages from spleen and heart and characterize M1 and M2 macrophage
polarization. In addition, we will look for IFN-G, IL-10 and IL-4 expression
levels on M1 and M2.
macrophages and try to
understand the immune response in Lyme disease. We will use cell specific
markers NOS2 and CD206 as representatives of M1 and M2 cells, respectively.
These markers will help us to identify phenotypes of M1 (F4/80 + NOS2) and M2
(F4/80 + CD206) macrophages within the joints and hearts of borrelia-infected
mice at a number of key time points. In addition to it, we will also
investigate the role of T cells in mediating borrelia arthritis and carditis.
We will study by using CD45+ and CD3+ markers for T cells and CD4+ for helper T
cells and CD8+ for cytotoxic T cells. We will also detect CD4+ T helper and
CD8+ cytotoxic T cell subsets by labeling with CD62L for naive, CD44+ for
effector and CD62L+ /CD44+ for memory T cells.
Immunophenotyping of T cells and macrophages
(i) Cell preparation:
Spleens and hearts will be
harvested from mice, placed in RPMI 1640 (Mediatech, Manassas, VA), and diced
with frozen slides in Hanks balanced salt solution medium (Cellgro, Manassas,
VA) to produce cell suspensions. Red blood cell lysis will be performed with
ACK lysing buffer (Life Technologies). Cells were then washed in RPMI 1640
containing 10% heat-inactivated fetal bovine serum (Atlanta Biotech) and 0.09%
sodium azide (Sigma-Aldrich, St. Louis, MO), followed by passage through 70-
m-pore-size and 40-m-pore-size nylon filters (BD Falcon, Bedford, MA).
(ii) Flow cytometric
cell staining:
Cell viability will be analyzed
by mixing splenocytes with acridine orange-propidium iodide staining solution
in a Luna-FL automated cell counter, and 3X106 cells per tube will be stained
as follows: cells will be incubated in 0.5 g Fc block (BD Biosciences) for 15
min at 4°C in staining buffer and incubated with the appropriate marker for
surface staining in the dark for 30 min at 4°C. The following surface markers
will be used: CD3 conjugated with fluorescein isothiocyanate (FITC), CD4
conjugated with phycoerythrin (PE), CD8 conjugated with allophycocyanin
(APC)-Cy7, CD62L conjugated with PE-Cy7, CD44 conjugated with APC,
CD45.2-peridinin chlorophyll protein [PerCP]-Cy5.5, NOS2-phycoerythrin [PE]),
F4/80-allophycocyanin [APC]-eF1.780, CD206-APC (all from Biolegend) and Pacific
blue as the Live/dead viability dye. For flow cytometry, cells will be acquired
on an LSR II flow cytometer (BD Immunocytometry Systems, San Jose, CA) equipped
with 405-nm, 488- nm, 561-nm, and 640-nm excitation lasers. Data will be
collected using BD FACSDiva software (BD Biosciences) and analyze using FlowJo
software (TreeStar, Ashland, OR). Fluorescence-minus-one (FMO) controls will be
used for gating analyses to distinguish positively stained from negatively
stained cell populations. Compensation will be performed using single-color
controls prepared from BD Comp beads (eBioscience) for cell surface staining.
Compensation matrices will be calculated and applied using FlowJo software
(TreeStar). Biexponential transformation is adjusted manually when necessary.
Cells will be gated on the basis of their forward and side scatter profiles.
Data analyses will be performed with acquisition of a minimum of 100,000
events.
Measurement of
inflammatory mediators in serum and cell-free supernatants of BMDM cultures:
The cytokine and chemokine
levels in the serum and cell-free supernatants of BMDM cultures will be
analyzed by mouse 38 plex kits. Mouse 38plex kits will be purchased from
eBiosciences/Affymetrix and used according to the manufacturer’s
recommendations. Each sample will be assayed in duplicate and
cytokine/chemokine standards and quality controls supplied by manufacturer will
be run on each plate. The multiplex ELISA will be repeated twice in two
different experiments. Data will be acquired by reading plates using a Luminex
200 instrument with a lower bound of 50 beads per sample per cytokine. Custom
assay control beads by Radix Biosolutions will be added to all wells. The
cytokine and chemokine response that shows statistical significance (P≤0.05) in
this study will be reported.
Quantitative real-time
PCR:
Total RNA will be isolated from
BMDM treated with live Bb and uninfected using RNA plus kit (qiagen). The
complementary DNA (cDNA) will be produced from
quality RNA (125 ng) samples
using an RT2 first strand kit as per the manufacturer’s instructions. The cDNA
will be analyzed for cytokines and chemokines that shows significance
difference in 38plex. The housekeeping genes, and negative controls will be
also used. Then the thermal cycling is performed (according to the
manufacturer’s protocol) and the real-time amplification data will be gathered
by using ABIPrism 7900HT software and analyzed.
Biotinylation of the
antibiotic for identification of its binding partners:
The primary amines of the the
antibiotic will be linked to N-hydroxysuccinimide ester of biotin like
NHS-LC-biotin (Pierce Chemical, Rockford, IL). The reaction mixture of fivefold
molar excess of NHS-LC-biotin and the antibiotic, 6-minopenicillanic acid, or
7-aminocephalosporanic acid will be dissolved in 0.1 M sodium phosphate buffer
and incubated for 30 min with gentle agitation. By incubating 30 min with a
primary amine like Affi-Gel 102 (Bio-Rad Laboratories) the reaction will be
terminated. The biotinylated antibiotic will be separated from the immobilized
ligand by centrifugation. Then the biotinylated antibiotic will be incubated
with borrelia cell lysates and pull down with streptavidin beads. The
biotinylated antibiotic-protein complex binding to streptavidin beads will be
eluted and analyzed with mass spectrometry for the specific protein sequences.
Preparation of oral
formulation of the antibiotic and tablets:
Microbeads of the antibiotic
will be prepared by ionotropic-external gelation technique. Beads of sodium
alginate containing unique co-polymers will be prepared using calcium chloride
as crosslinking agent. The antibiotic will be mixed with 1% of CMC and 2% of
sodium alginate and mixed until it is dissolved. Then the mixture will be
extruded drop wise by using 26 G syringe into 2% calcium chloride solution to
produce sodium alginate beads. The antibiotic entrapped alginate beads will be
cured for 30 min and centrifuged. Then the antibiotic alginate beads will be
enteric coated with 5% eudragit-S100 for 30 min. Finally, the antibiotic beads
will be dried overnight. Then the enteric coated antibiotic beads will be
packed in the gelatin capsules and administered to rats orally. After oral
administration of antibiotic beads to Sprague Dawley rats, blood will be
collected from tail vein at 15min, 30 min, 1h, 2h, 4h, 8h, 16h and 24 h for
analyses by mass spectrometry.
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