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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