Radiol Oncol 2000; 34(1): 59-65. Influence of hydralazine on interstitial fluid pressure in experimental tumors - a preliminary study Blaž Podobnik and Damijan Miklavčič Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia Background. Interstitial fluid pressure (IFP) has been recognised as the mostimportant obstacle in macro-molecular drug delivery to solid tumors. Our interest was to reduce differentialy tumor IFP with respect to IFP in surrounding and normal tissues in order to increase drug delivery to tumors as well to increase tumor blood flow and potentialy tumor tissue oxygenation. In this preliminary study we used hydralazine, a long-acting arterial vasodilator. Materials and methods. Measurements ofinterstitial fluid pressure were performed in vivo on CBA mice bearing SAF tumors using wick-in-needle technique. Altogether eleven measurements were obtained on different animals with tumors of different size. Results. IFP in tumors after hydralazine administration was significantly lower than initial values in corresponding tumors. On average tumor IFP decreased far 33 % from initial value. On the contrary, no change in IFP in normal tissue was observed after hydralazine administration. Also, after injection ofphysiological saline instead ofhydralazine there was no change in IFP neither in tumors nor in muscle. The results ofour preliminary study on the effect of hydralazine on IFP in SAF tumor model is in accordance to previously reported studies. The decrease in tumor IFP was only observed in tumors, but not in muscle and surrounding subcutis. Conclusion. Hydralazine is a vasodilator which is capable ofdecreasing tumor IFP, reproducibly and with favorably long lasting dynamics. Key words: sarcoma; experimental-drug therapy; hydralazine; extracellular space; interstitial fluid pressure; manometry Introduction Interstitial fluid pressure (IFP) has been recognised as the most important obstacle in macromolecular drug delivery to solid tu-mors.1,2,3 IFP was also correlated with tumor Corespondence to: Damijan Miklavčič, DSc, Faculty of Electrical Engineering, Tržaška 25, 1000 Ljubljana. Tel: +386 61 176 84 56; Fax: +386 61 126 46 58; E-mail: damijan@svarun.fe.uni-lj.si blod flow.4,5 Recent clinical study involving patients with cervical carcinoma6 reported that tumors with high IFP were more likely to be hypoxic and less likely to regress completely after radiotherapy. Elevated IFP in solid tumors hinders fluid filtration from tumor vasculature which is the prime driving force for macromolecular transvascular flow. However, it is not clear at the moment how elevated IFP would affect tumor blood flow 60 Podobnik B et al. / IFP in experimental tumors and oxygenation. Nevertheless, there is an obvious and increasing interest in modulating IFP in solid tumors thus by decresing it, facilitating macromolecular drug delivery like monoclonal antibodies into tumor tissue and possibly modyfing tumor blood flow and tissue oxygenation. Various vasoactive drugs have been used with variable success to modulate solid tumor IFP. Most of the drugs used (e.g. nikotinamide, angiotensin II, epineph-rine, norepinephrin, nitroglycerin and hydra-lazine) have been reported to modulate tumor IFP.7 In general, vasoconstricting agents resulted in increase of tumor IFP whereas vasodilating agents produced decrease in tumor IFP. Our interest was to reduce differen-tialy tumor IFP with respect to IFP in surrounding and normal tissues in order to increase drug delivery to tumors as well to increase tumor blood flow and potentialy tumor tissue oxygenation. In this preliminary study we used hydralazine, a long-acting arterial vasodilator. After i.v. administration we measured IFP in solid subcutaneous tumors (SAF anaplastic sarcoma) and subcutis close to tumor and/or muscle tissue in CBA mice. Anesthesia The treatment and measurements of animals which could cause discomfort or pain to animals were performed under general anesthesia. The mice were anesthetized with Isoflurane (Flurane-Isoflurane, Abbot Labs Ltd., UK; waporizer-Isotec 337C, Ohmeda, USA) gas anesthesia (1.5-2% of Isoflurane was mixed with NO2, 02 mixture; flow of NO2 and O2 was 0.6 1/min). Animals were anes-thesied and placed on a heating pad (TCU 035,27S, Cheshire, UK) which maintained stable body temperature. Throughout the experiment rectal temperature and heating pad surface temperature were monitored. The rectal temperature was between 37 and 38 °C and the maximal surface temperature of heating pad was lower than 40 °C. Drugs Hydralazine (Hydrazinophthalazine, Sigma Chemical Co., St. Louis, MO) was dissolved in sterile saline (0.9% NaCl) prior to each experiment. A dose of 2.5 mg/kg was injected intravenously (i.v.) into retroorbital sinus. Materials and methods Animals and humor model All experiments were performed on 8 to 10 week old female CBA mice which were maintained under standard laboratory conditions with food and water ad libitum. The SAF (anaplastic sarcoma; 0.1 ml of crude tumor cell suspension) was transplanted subcutaneous-ly under sterile conditions dorsolaterally on a right flank of the mice. Experiments were performed on tumors of different size ranging from 95 mm3 to 800 mm3. All experiments were performed at the Department of Tumor Biology, Institute of Oncology, Ljubljana in accordance with ethical provisions for research on animals. Measuring technique and experimental protocol IFP was measured by the wick-in-needle tech-nique8'3'9 using a 0.5 mm (25G) needle probe (Terumo Belgium) with a 2 mm sidehole about 3 mm from the tip. The needles were filled with two surgical thread fibers (5-0, Seide Silk). Prior to each experiment the measurement system was calibrated. All recordings of IFP were performed as two channel measurements, measuring IFP in tumor and in subcutis close to tumor or muscle. Needles were connected to pressure transducers (TSD104 and TSD104A, Biopac Systems Inc., CA-Goleta, USA) by a polyethylene tube and the entire system was filed with physiological saline (0.9% NaCl) which contained heparin (Krka, Slovenia) 72 u/ml for preventing blood clots to be formed. Special care was taken to Radiol Oncol 2000; 34(1): 59-65. 61 Podobnik B et al. / IFP in experimental tumors avoid trapping of air bubbles in a system during the filling. Saline in system was used as a conductor of pressure. Pressure transducers were connected via amplifier (DAlOOA, Biopac System Inc.) and data acquisition unit (MPlOO, Biopac Systems Inc.) on personal computer. The sampling frequency was 10 Hz. During calibration of a measurement system zero reference pressure was obtained by placing the needles in a heparinesed physiological saline-filled beaker and calibration of pressure was done by elevating or lowering the beaker. At different levels (1cm and 30 cm) of liquid column (level O cm was equal to level of needle insertion into a tumor or muscle), output voltages of pressure sensors were measured and used for calibration. After the calibration one needle was inserted into the center of a tumor and the other one into subcutis or a muscle of a right hind limb. After that needles were slightly withdrawn to avoid compression of the tumor and muscle under the probe tip and left in place without external fixation. All measurements lasted for 2 hours or longer. One of complete IFP measurements is given in Figure 1. After the initial equilibration period, compression/decompression (C/D) test was performed. This test allowed us to verify the continuum between the fluid phase in interstitium and needle lumen. By tightening the clamp on a polyethylene tube so as to inject a volume of approximately 0.2 into the tissue caused a sudden pressure rise (Figure 1). The pressure then declined, first rapidly and then more slowly, restabi-lized to the initial level within 30 seconds to 2 min. Withdrawal of the same amount of fluid by loosing the clamp gave a reverse response, a sudden fall in pressure with gradual return to the original level (Figure 1). This test was performed at the beginning and at the end of compresion (C) TUMOR , j dec°mpresion (D) injection ofHYZ ........I C 30.00 -15.00 0.00 50.00 100.00 tirne (min) 150.00 Figure l. An example of IFP recording during the entire experiment. IFP in tumor and muscle tissue including compression (C) /decompression (D) test, injection of hydralazine (HYZ) and calibration chack are given. Radiol Oncol 2000; 34(1): 59-65. 62 Podobnik B et al. / IFP in experimental tumors each experiment and gave us the information about the quality and reliability of IFP measurements. Only results obtained in experiments where both tests were correct, were accepted and considered as reliable. Hydralazine was injected i.v. after a stable recording of IFP was obtained. The response of IFP to hydralazine was monitored for approximately one hour. After that period C/D test was performed again and needles removed from tissue. Measurement was finished with the calibration test in order to verify the calibration procedure performed prior to the beginning of the experiment.10 Data processing and statistical analysis Initial IFP values in tumors and muscle or subcutis was determined as the mean value of IFP recording in the interval of app. 20 minutes duration after the first C/D test and prior to hydralazine injection. The values of IFP after hydralazine injection were determined as the mean value of IFP recording in the interval of app. 30 minutes duration starting at 10-15 minutes after hydralazine (or physiological saline) injection. All values are reported in tables and figures as mean±standard deviation. Statistical analysis of the data was performed with a paired t-test comparing measured values of IFP values in tumors before and after hydralazine injection, IFP in muscle tissue or subcutis before and after hydralazine injection after normality test was performed and fulfilled. Exact p-values are reported. 18 16 14 ^^ , „ o 12