Feasibility of Rapid Mitoxantrone-DNA Binding Revealed by Diffuse Reflectance Spectroscopy

Singh-Moon R*, Bigio I#, Joshi S*. *College of Physicians and Surgeons, Columbia University, New York, NY, USA. #Boston University, Boston, MA, USA .

Introduction

Discussion

Reliable optical measurements of drug concentrations require that the optical properties of drug or tracer remain stable during measurements. However the optical can change when injected in-vivo due to tissue binding or metabolism. We have used mitoxantrone extensively for our optical measurements and in tracking drug delivery assuming that the spectrum of the drug is stable and the metabolism over short period of time after injection is negligible. Hitherto these measurements were based on in-vitro characterization of the absorption spectrum. Our recent observations using diffuse reflectance spectroscopy (DRS) in-vivo suggest that a subtle shift in MTO spectrum occurs soon after injection. We hypothesized that such a shift is possible due binding with the DNA. • Mitoxantrone (MTO) has a light absorption in the visible-near infrared region that can be observed with diffuse reflectance spectroscopy.

•  In published literature, MTO experiences a spectral shift in ms time frame when bound to albumin (8 nm) and an additional 7–15 nm to nucleic acids, which is reported to take much longer.

• Diffuse reflectance spectroscopy (DRS) can determine changes in tissue spectra with sub-second time frame and can perimit detection of spectral shifts after MTO injections.

• We therefore assessed the feasibility of DRS for simultaneous determination of tissue albumin- and DNA-bound MTO concentrations after IV injection.

•  A DRS-based method for determining MTO-DNA binding could assist in non-invasive monitoring of delivery to the target site. Instead of adding to the noise in measurements such changes could provide novel insights into drug kinetics that are not be easy to asses by conventional pharmacokinetic tools.

Results

Methods

Fib 2: Clearance of Albumin bound MTO and increase in DNA-MTO

• After IACUC approval, experiments were conducted in Sprague Dawley rats under isoflurane anesthesia.

• Tissue drug concentrations were measured from the animals’ heel by DRS.

• MTO (1mg/1 min) was infused via the tail vein to keep systemic hemodynamics stable. After 12 baseline measurements 600 spectra were obtained over the next hour.

• The concentrations of MTO states were determined using extinction spectra for albumin and DNA-bound MTO and oxy-and deoxy-hemoglobin obtained from published literature.

•  Finally, drug concentration-time curves were fit to a 2-compartment model for IV injection using least squares minimization in conjunction with a Runge-Kutta numerical solver.

Acknowledgment: NIH R01(s): CA 127500 and CA 136843 (S.J.).

Conclusions •  This experiment shows that it is feasible to sometime detect spectral shifts

consistent with MTO binding to DNA - which is the target site of drug delivery and is therefore significant.

•  Further studies are necessary to validate actual tissue uptake by determining MTO concentrations in cell fractions.

•  An optical method monitoring chemotherapeutic drug-DNA concentrations could provide a parameter to assess the effectiveness drug delivery particularly when the drug is caged in nanoparticles.

•  One potential problem with nanoparticle drug delivery is the suboptimal release of the drug and therefore interaction of drugs with their molecular targets is of great significance.

•  Chemical tissue concentrations usually do not provide such information, while optical methods might easily be able to do.

Fig 1: Shift in mitoxantrone (MIX) spectrum due to DNA binding. From: Agarwal S et al: J of Photochemistry and Photobiology B

estimated from the ratio of the intercept to the slope[11,15,30,39,44] (Fig. 6). The binding constant for this interactionis 3.8 ! 105 M"1, which indicates that mitoxantrone has a highaffinity with DNA double helix. The value of binding constant ob-tained is consistent with those reported for the interaction ofanthracycline molecules with DNA [36–40].

4. Conclusion

In this study, we investigated the interaction of anticancer drugmitoxantrone with calf thymus DNA using spectroscopic tech-niques. Several spectral changes were observed after the bindingof mitoxantrone with DNA. Results affirmed that mitoxantroneintercalates between the base pair of DNA. Spectroscopic resultsindicate that mitoxantrone binds with guanine (N7), thymine(O2) and cytosine (O2) sites. Mitoxantrone also binds externallywith the phosphate–sugar backbone of DNA double helix throughits side chains. There is no major change in the conformation ofDNA, although slight perturbations in the DNA double helix confor-mation occur upon mitoxantrone interaction.

Acknowledgements

The authors thank Director, CSIR–National Physical Laboratoryfor granting the permission for publication of the work. S.A. isthankful to Council of Scientific & Industrial Research (No. 31/001(0373)/2011-EMR-I) and D.K.J. is thankful to Indian Council ofMedical Research (No. 3/1/ JRF/8/MPD/2007. 31849) for financialsupport as junior research fellow.

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Fig. 5. UV–Visible spectra of free mitoxantrone (1 ! 10"2 mM) and mitoxantrone–DNA complex.

Fig. 6. Double reciprocal plot of mitoxantrone binding to DNA. A0 is the initialabsorption of free DNA and A is the absorption at different concentrations at260 nm. C is the analytical concentration of mitoxantrone in solution.

S. Agarwal et al. / Journal of Photochemistry and Photobiology B: Biology xxx (2012) xxx–xxx 5

Please cite this article in press as: S. Agarwal et al., Spectroscopic studies of the effects of anticancer drug mitoxantrone interaction with calf-thymus DNA,J. Photochem. Photobiol. B: Biol. (2012), http://dx.doi.org/10.1016/j.jphotobiol.2012.11.001

• Representative curves showing concentration-time clearance of albumin and DNA bound MTO are shown in Fig 2.

•  Rapid increase in albumin bound MTO concentrations was evident immediately after injection, and binding to DNA was evident early after injection.

• While concentrations of albumin-bound MTO declined below the level of DRS detection, those of DNA-bound MTO increased slowly but remained stable thereafter.

• The pharmacokinetic data for albumin and DNA bound MTO was in good fit with the 2-compartmental model in two of the five animals.

•  It is unclear as to the significance of the observed shift in peak absorption after MTO injection is unclear as yet:

•  First, the shiftt was not consistently obesrved.

•  Second, in previous studies in tumor bearing animals when a 13 nm shift in spectrum was factored in there was better curve fitting on experimental data. However thi shift was much delayed and did not complete until 70 minutes.

•  Third we have also observed a subtle shift in MTO spectrum in vitro after dissolving the drug in propofol which was used in our animals to supplement anesthesia.

•  However, what is clear from these experiments is that there is a subtle shift spectral properties of the compound soon after injection.