Our Sponsored Research

---

Fluoroquinolones  are a group of antibiotics that are widely prescribed to treat a  variety of bacterial infections. While the intended target is bacteria,  the fluoroquinolones are also known to interact with the human genomic  system by conjugating with Deoxyribonucleic acid (DNA). Given the  crucial role of DNA in human body function, any damage may have serious health consequences.   

The study we organized was conducted with subjects suffering from Fluoroquinolone Associated Disability (“FQAD”): 

Objective:

Determine if conjugates form between Levofloxacin, Ciprofloxacin genomic and mitochondria DNA.

Study Parameters:

(A1). Study for fluoroquinolone DNA adduct. Human blood samples [50+ participants/samples from suspected cases FQAD] 

(A2). Study for fluoroquinolone mtDNA adduct. Human blood samples [50+ participants/samples from suspected cases FQAD] 

(A3).  Study for fluoroquinolone genomic DNA adduct. Human blood samples [50+  participants from suspected cases FQAD, plus samples/4 children who had  not taken a fluoroquinolone, but their mothers did before or during  pregnancy] 

(A4). Control group who had never taken a fluoroquinolone
 

Study Design:
-description of fluoroquinolone DNA adduct
- methodology and strategy of study
- standard spectrogram fluoroquinolone only
- spectrogram of control blood sample

 -spectrograms representing the in vitro study for FQ DNA adduct after exposure of control blood to fluoroquinolones
- spectrograms of fluoroquinolone adduct to genomic and mitochondrial DNA
- diagrams showing the mechanism of fluoroquinolone adduct to DNA

- summary, test results and substantiation
 

In  the link below, you will find a sample of the results on the Levofloxacin adduct to mitochondria and genomic DNA along with methods used during this study: 

https://www.dropbox.com/s/2xjflceh39jbvtz/FQ%20DNA%20ADDUCT%2058%20JT%20HIPAA.pdf?dl=0

Key Findings:

Analysis of Human Genomic and Mitochondrial DNA was made by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS / MS) for presence of adducts based on test for genomic FQ DNA adduct when exposed to Levofloxacin (abstract from FQ DNA adduct test result).

Introduction:

Fluoroquinolones  are a group of antibiotics that were widely prescribed to treat a  variety of bacterial infections. While the intended target is bacteria,  the fluoroquinolines were also known to interact with human genomic  system by conjugating with Deoxyribonucleic acid (DNA). There were also  numerous aberrations caused at various body tissue levels. Given the  crucial role of DNA in human body function, any damage to it has serious  health consequences. The objective of the present analysis of the  patient's blood is to see if any conjugates (adducts) are formed between  Levofloxacin and DNA.  The data presented here is limited to one patient and is subject to confirmation by a more elaborate investigation.

Materials and Methods:

Commercially  available kits from Sigma-Aldrich Company (Kit No. NA2020) and from  Quiagen Inc. (Kit No. 37612) were employed for isolation and  purification of DNA, using the manufacturer's standard procedures.

Experimental Strategy:

A.  Create an Adduct Molecule out of a reaction between Control Blood and  Levofloxacin, and assign a chemical structure to the Adduct.

B. Analyze the Patient Blood for the presence of the Adduct and / or Levofloxacin.

C.  If the Adduct molecule did not appear intact but appeared in a  modified form, examine possible relationship to the precursors.

Results:

Reference  Standard of Levofloxacin yielded it characteristic spectrum with its  molecular ion of m / z 362, and major daughter ion of m / z 342/344  (343) (Figure 1). Analysis of Control Blood

(Untreated  with Levofloxacin) provided the background ions (Figure 2). Control  Blood treated with Levofloxacin showed a product of m / z 494 (Figure  3). When the ion of m / z 494 was analyzed for its daughter ions, an ion  of m / z 343 was obtained (Figure 4). This ion of m / z 343 was a  common ion between Levofloxacin and the Adduct, thereby establishing the  relation between these 2 molecules. HPLC-MS analysis of Patient's  Genomic DNA showed several ions that were related to Levofloxacin  (Figure 5). Comparable data was also seen with Patient's Mitochondrial  DNA (Figure 6). Also, similar results were obtained with the 2 samples  after acidic hydrolysis (Figures 7 and 8).

Discussion:

FDA  Label of Levofloxacin states that:      "Levofloxacin is mainly bound to  serum albumin in humans."  "Levofloxacin undergoes limited metabolism in  humans and is primarily excreted as unchanged drug in the urine.  Following oral administration, approximately 87% of an administered dose  was recovered as unchanged drug in urine within 48 hours, whereas less  than 4% of the dose was recovered in feces in 72 hours. Less than 5% of  an administered dose was recovered in the urine as the desmethyl and  N-oxide metabolites, the only metabolites identified in humans. These  metabolites have little relevant pharmacological activity. "

The  above situation needs to be reconciled with the general belief that  certain toxic compounds may be stored in the adipose tissue and released  later. The non-target effects of Levofloxacin may, therefore, be either of one time occurrence (immediately after the dosage), or of a minor  recurrence (from storage). Given the pharmacological potency of  Levofloxacin, its total elimination from patient's tissues may be  important in assessing persistent disease condition.

Conclusions:

An  Adduct Molecule could be formed between Levofloxacin and Control Blood.  The Adduct was structurally characterized, using Mass Spectrometry  (HPLC-MS / MS). Although the exact configuration of the Adduct was not  obvious in the Patient's DNA, the ions lend support to its origin to be  Levofloxacin and Guanine (a portion of DNA molecule).

A proposed pathway towards the formation of the Adduct molecule is shown in Figures 9 and 10.

In  tests with Ciprofloxacin in vivo tests, the mechanism of adduct  formation between genomic and mitochondrial DNA was identical to that of  Levofloxacin. The same components of guanine and the antibiotic,  Ciprofloxacin, and major daughter ion were found.  The  crowning achievement of the research were the in vivo tests which overlapped with in vitro tests. All mtDNA genomic DNA patients  previously exposed to Ciprofloxacin and Levofloxacin developed identical  adducts as in the in vitro study. In  the case of the children, it is necessary to perform tests on a larger group because the adduct also occurred in their case, even though they  had never been treated with  these antibiotics,  but their mothers did before or during pregnancy.  Another  important observation was that the adduct may be formed regardless of  the dose, form of its intake, age, brand and gender.

We  know that other laboratories in the world have performed individual  tests on FQ DNA adduct in humans, in various laboratories conducted in  2010 and 2019, 2020, 2021. Their results are in line with the research  organized by our foundation. One laboratory has found that some  individuals maintain fluoroquinolone in lymphocytes even after 9 months  of exposure. This phenomenon of accumulation and DNA adducts by  fluoroquinolones and mitochondrial damage is perceived by us as a  serious global problem that poses a threat to the next  generation.

*** For more information on adducts go to "Adduction" in our library. 

This study was performed utilizing a representative group of people, injured after treatment with fluoroquinolones, experiencing Fluoroquinolone Associated Disability (“FQAD”):  

Objective:

To search for future FQAD therapies using iPSC technology.

 Study Design:

-Isolation of epithelial cells from urine samples from patients  

-Culture and preparation of urinary epithelial cells for the reprogramming process 

-Reprogramming of epithelial cells from urine into induced pluripotent stem cells (iPSc)  

-Stabilization of obtained iPSc colonies, identification of pluripotency markers (SOX2, Oct3 / 4, TRA-1-81, TRA-1-60)  

-Banking the obtained iPSc lines 

-Differentiation of the iPSc line to neuronal stem cells (NSC)  

-Stabilization of the obtained NSC lines, identification of markers (Nestyna, SOX2)  

-Banking of NSC lines obtained

____________________________________________________

Complete & Published May 21, 2022      

Regeneration difficulties in patients with FQAD can limit the use of iPSc-based cell therapy.       

Dagmara Grot, Katarzyna Wasiak, Jerzy Tyszkowski, Ewelina Stoczynska-Fidelus, Tomasz P. Ochedalski & Piotr Rieske 

Stem Cell Research & Therapy volume 13, Article number: 210 (2022) 

https://stemcellres.biomedcentral.com/articles/10.1186/s13287-022-02886-0?fbclid=IwAR1k1sKTMNXQ7VU4BcBFXJy7SINvDIAtcuUUpo5IICtv4f7RQCvhbrQ8xTc#Sec2       

Important abstracts from publication:

Study Parameters: 

1). 10 Fluoroquinolone exposed subjects in age range 20-79 years  

2). Fluoroquinolone exposed subjects time frame of .5 years to 10 years since prescription completion 

3). Exposed subjects with both immediate and delayed drug reaction 

4). Exposed subjects who had been prescribed either Levofloxacin or Ciprofloxacin in pill or otic forms 

5). Exposed subjects who were having consistent and current side effects 

6). Exposed subjects who were in good health before Fluoroquinolones 

7). Control group who had never taken a Fluorqouinolone 

Introduction: 

Quinolone antibiotics kill bacteria by inhibiting enzymes called class II topoisomerases. These enzymes are involved in untangling DNA during cell proliferation. Quinolones bind to these enzymes, thus preventing normal enzyme reactions [1]. In the 1980s, researchers modified quinolones by adding fluorine atoms to the compound structure, increasing these antibiotics penetrance into tissues. Penetrated tissues include the central nervous system and cardiac tissues, which improve effectiveness against bacterial infections. These actions, however, also caused death and damage to organs such as the liver. Therefore, some FDA-approved fluoroquinolones (FQ) were withdrawn from use [2]. Still, many patients suffer after using approved antibiotics, developing enigmatic and quite a severe spectrum of side effects, finally classified by the FDA as fluoroquinolone-associated disability (FQAD) [3]. In the case of FQs, it is suspected that symptoms are caused by mitochondrial [4, 5] and genomic DNA [6,7,8] damage. To this end, we attempted to develop an induced pluripotent stem cell (iPSc) model to study this disease and verify whether reprogramming technology can be used in the future to treat patients with FQAD and their other disorders in which autologous-induced pluripotent stem cells and their derivatives may be used. Urinary cells are considered as relatively easy to reprogram [9]; unfortunately, iPS cells could not be easily generated from these patients’ somatic cells. This raises additional concern about global FQ use and the accessibility to iPSc-derived treatments for FQAD patients.   

Results:

 For three donors (DONOR 1–3), stable urine primary cell cultures could not be obtained. The isolates from another four out of ten donors (DONOR 4–7) contained single viable epithelial cells which became senescent very quickly. Two of the stable primary cell cultures (DONOR 8 and DONOR 10) became senescent right after transfection with reprogramming episomes (Fig. 1b). Finally, urinary epithelial cell cultures derived from three out of ten individuals with FQAD were suitable for being subjected to the process of reprogramming. Only one donor provided cells that were successfully reprogrammed. During parallel studies conducted with healthy donors, success was achieved in six out of ten cases [9].  

Discussion: 

The effect of fluoroquinolones on cells and tissues is poorly understood. IPS cells have become a valuable research model for many diseases. In the case of FQs, it is suspected that symptoms are caused by mitochondrial and genomic DNA damage [4,5,6,7,8]. The influence of these changes on cells can be tested, with the use of an iPS cells model. IPS cells may also become a potential therapeutic tool for patients with FQAD. Starting from iPSc, regenerative therapy could be carried out, beginning with the typical tendon damages found with FQAD. In the case of mitochondrial damage, it is worth considering the selection of iPS cells with the highest percentage of the normal mitochondria for therapeutic purposes. Such an approach could possibly allow for the selection of suitable cells to develop advanced therapeutic medicinal products.  Urine cells are well known to be very accessible and easy to reprogram. Drozd et al. [9] showed that these cells generate a higher number of iPSc colonies in comparison with skin cells (urine cells are 100 times more efficient). Scar cells were the most difficult to reprogram (about 500 times less efficient than urine cells), which could have been an issue to use in this study as FQAD patients frequently show structural skin damage. According to Drozd, et al., epithelial phenotypes of urine cells are most likely pro-reprogramming. It is well known that fibroblastic, but not epithelial cells, must go through MET during reprogramming. Finally, the subpopulation of urine cells shows TRA-1-60 and TRA-1-81 expression [9]. The reprogramming efficiency of blood cells is similar to fibroblast reprogramming efficiency [11]. All the above suggests that other cells can be more difficult to reprogram than urine cells when it comes to cases of FQAD; however, we cannot exclude some unique damage to the kidney in this syndrome. ROS analysis showed no differences between cells from healthy donors and cells from FQAD patients, suggesting that oxidative stress, in this case, is not directly related to cell senescence and failure of reprogramming.  This study showed that it is very difficult to generate iPS cells from urine epithelial cells of patients with FQAD. Equally important is the fact that the efficacy of the cell culture establishment was very low, with only one out of ten patients providing cells suitable for reprogramming. This is surprising as other previous studies showed that urine cells should be a very efficient source for reprogramming [9, 12]. An important fact about FQAD patients is that their connective tissue is damaged; however, this study also suggests that renal structures can be preferentially damaged by FQ’s. It has to be emphasized that so far, no one has been able to define which exact type of cells from urine become reprogrammed; however, in our previous research we detected cells showing markers for stem cells [9]. Verification of the presence and the percentage of these cells in urine from FQAD individuals should be considered. If that premise is accurate, it would serve as a marker for the malfunctioning of regenerative systems.  It seems that there is no easy model of cell reprogramming when studying this syndrome, which might limit research opportunities. Future efforts to apply regenerative medicine to FQAD individuals based on reprogramming technology will be a challenging process that will need to be refined. The fact that this study was able to establish the first model of an iPS cell line from a person with FQAD may provide hope for the creation of future treatments. 

Study was organized to test the cytotoxic activity of Fluoroquinolones based on the determination of the IC50 (inhibitory concentration), as well as IC25 and IC10 values; for comparative purposes, streptomycin and tetracycline were included. For this purpose, endoderm cells, neurons, cardiomyocytes and osteogenic cells in a wide range of concentrations will be tested. This study will be performed utilizing a representative group of people, injured after treatment with fluoroquinolones, experiencing Fluoroquinolone Associated Disability (“FQAD”):  

Objective:

To  utilize the MTS test to assess the cytotoxic activity of fluoroquinolones.