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The effects of single dose oral hydralazine on blood flow through human lung tumours
Radiotherapy Elsevier RADION
and Oncology,
18 (1990) 283-292
283
00742
The effects of single dose oral hydralazine on blood flow through human lung tumours N . P . Rowe11 1,4, M. A. Flower3, V. R. McCready2, ‘Academic
B. Cronin* and A. Horwich’
Unit, Department of Radiotherapy, Departments of 2Nuclear Medicine, and 3Physics as Applied to Medicine, Royal
Marsden Hospital, Sutton, Surrey ,942 5PT, U.K., and 4MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD, U.K.
(Received 24 May 1989, revision received 22 January
Key words: Hydralazine;
1990, accepted
Blood flow; Bronchial carcinoma;
15 February
99Tcm-HMPAO;
1990)
SPECT
Summary Hydralazine has been shown to reduce tumour blood flow and to potentiate the cytotoxicity of melphalan and bioreductive agents in mice. In order to determine whether such a strategy might have clinical potential, a study was undertaken to investigate the effects of hydralazine on blood flow through human tumours. Twenty-two patients with carcinoma of the bronchus received a single oral dose of hydralazine in the range 25 to 150 mg (0.37-2.86 mg/kg) according to age and acetylator status. Tumour blood flow was assessed by single photon emission computed tomography (SPECT) performed 10 min following intravenous 99Tc”-HMPA0 on two occasions 2-8 days apart, the second being performed 60 min after hydralazine administration. In 20 evaluable patients, hydralazine caused a 38% increase in blood flow through the whole tumour ( p = 0.007) and a 28 % increase in flow through the tumour centre (p = 0.03) with greater increases occurring in patients sustaining greater falls in peripheral resistance. Tumour vascular resistance fell indicating active vasodilation in arterioles supplying tumours. Side-effects due to hydralazine were reported by eight patients.
Introduction The blood supply of a tumour may be important both in its natural history, influencing metabolism and growth, and in determining response to treatment. Early investigations into the effects of vasoactive agents on tumour blood flow were unAddress for correspondence:
Dr. N. P. Rowell, Department of Radiotherapy and Oncology, University College Hospital, Gower Street, London WClE 6AU, U.K. 0167-8140/90/$03.50
0 1990 Elsevier Science Publishers
dertaken in order to identify drugs which, by increasing tumour blood flow (and possibly also reducing flow through normal tissues), might enhance the effects of radiation. Although factors governing blood flow through tumours are now better understood [ 181 and the effects of vasodilators on tumour blood flow have been extensively reviewed [ 191, full interpretation of reported studies is only possible when physiological parameters (i.e. blood pressure and cardiac output) have been recorded. Studies which report
B.V. (Biomedical
Division)
284 tumour blood flow in terms of fractional distribution of cardiac output (FDCO) are difficult to interpret because of the increase in cardiac output produced by almost all vasodilators. In this study the term “blood flow” refers always to nutritive perfusion; AV shunting is not assessed. As a result of early studies, Cater [ 51 concluded that blood flow through tumours varied with systemic blood pressure but with the notable exception of the vasoactive amines (e.g. noradrenaline and acetylcholine) which appeared to have direct effects on tumour vessels. These direct effects have been demonstrated by others with vasoactive amines [ 16,221 and more recently with calcium channel blockers [23,24,43,48]. There is now abundant evidence that in spite of the histological appearance of scanty smooth muscle associated with tumour vessels [6], vessels may constrict or dilate in response to drugs and that blood pressure is not the sole determinant of tumour blood flow. Studies with the vasodilator hydralazine have variously reported no effect on tumour blood flow (0.5-1.0 mg/kg iv. in anaesthetised rats) [ 1,6], a 50% decrease (10 mg/kg i.p. in anaesthetised mice) [ 151, a 54-90% decrease (0.5 mg/kg i.v. in anaesthetised dogs) [ 1,441 and a 67% decrease (10 mg/kg i.v. in anaesthetised mice) [42]. Although these results appeared to suggest that hydralazine and calcium-channel blockers produced qualitatively different effects on tumour blood flow, the possibility that the two end-results were simply due to differences in dose could not be excluded. Hydralazine has been available as an antihypertensive for over 30 years and dose-response relationships and pharmacokinetics are well understood [26,36]. Oral medication was chosen for this study because of occasional reports of stroke and myocardial infarction following intravenous administration. In man, blood flow patterns have been investiand single photon gated with 99Tcm-HMPA0 emission computed. tomography (SPECT) in brain turnout-s [2,25], lung tumours [33], breast tumours (Rowell et al., unpublished data) and in tumours in a variety of sites [41]. Except for very
low values, tumour : lung ratios in repeat studies performed after an interval of 3-5 days were within k 15% of initial values [33]. r Studies with phantoms have been undertaken to develop a method for correction of SPECT data so that perfusion in a particular region of interest may be expressed quantitatively (Rowell, unpublished data). The difficulties in relating counts/pixel in reconstructed images to absolute activity in tissue are due principally to the effects of scatter and the inadequacy of attenuation correction [ 12,17,20,46]. Discrepancies are greater for smaller and more centrally placed sources. For this study, primary lung tumours were chosen because in SPECT images most were surrounded by a relatively homogeneous background, a situation easier to simulate in phantom studies. In contrast to metastases, primary lung tumours derive almost all of their blood supply from the systemic circulation [28] so that the amount of a radiopharmaceutical retained in a tumour can be expressed as a fraction of that delivered to the systemic circulation (i.e. FDCO). A reduction in tumour blood flow might be turned to therapeutic advantage as hydralazine potentiates the cytotoxicity of melphalan [40], misonidazole [ 141 and the bioreductive agents RSU 1069 [8,14] and SR4233 [4] in mice. Bioreductive drugs are those which undergo selective activation in hypoxic cells by redox cycling [ 381. Administered to mice postirradiation, hydralazine enhances the effectiveness of misonidazole and RSU1069 in transplantable murine tumours [9,39] but not in human tumour xenografts [ 141. In order to assess whether blood flow reductions might be achieved at clinically tolerable doses, we elected to study the effects of single dose oral hydralazine on blood flow through primary lung tumours.
Patients and methods Twenty-two patients with carcinoma of the bronchus (18 men, 4 women; mean age 66.9 years, range 52 to 74) were studied. All patients with a
285 diagnosis of carcinoma of the bronchus under the age of 75 years were eligible provided that on chest radiographs tumours could be clearly defined and there were no adjacent areas of collapse or consolidation. Patients were excluded if there was a history of stroke or myocardial infarction or if there was evidence of uncontrolled hypertension, uncontrolled cardiac failure or raised intracranial pressure [ 301. Patients with atria1 fibrillation or valvular heart disease were excluded because of greater errors in the estimation of cardiac output. The study was approved by the Royal Marsden Hospital Ethics Committee and informed consent was obtained from each patient. Following initial assessment and bronchoscopy by the referring physician, histological diagnosis remained uncertain in three patients despite a confident clinical diagnosis. Of the remainder, fourteen had squamous carcinoma, two small-cell carcinoma, two adenocarcinoma and one a poorly differentiated carcinoma of uncertain type. In two patients, there were two tumour masses on chest radiograph and CT. At the time of the study, 14 patients, were undergoing investigations prior to radiotherapy and three prior to surgery; five with advanced but asymptomatic disease remained under observation. No patient had received prior radiotherapy. Tumour size was assessed from the chest radiograph. Two perpendicular diameters (X,Y) were measured directly from the PA view and demagnified. Where lateral views were not available or were unhelpful, the third dimension (Z) was calculated as the mean of X and Y. Magnification factors of 1.05 for PA views and 1.08 for lateral views were assumed. Tumour volume was calculated as : Tumour volume = T *(X x Y x Z) Equivalent diameter (assuming a spherical configuration) for use in the correction of SPECT data was then calculated using the same formula (mean 5.5 cm; range 2.3 to 9.3 cm). Radial position was measured from CT scans or, when not available, from SPECT images.
The study required out-patient attendance for two afternoons. Both afternoons began with the patient resting on a couch for 30 min. At the end of this 30-min period on the first afternoon, 400 MBq 99Tc”-HMPA0 (Ceretec, Amersham International plc) was administered intravenously and on the second afternoon, hydralazine tablets (Apresoline, Ciba-Geigy) were given by mouth and 400 MBq 99Tc”-HMPA0 administered 60 min thereafter. Blood pressure and cardiac output were measured noninvasively [ 341 during and 5 min after 99Tc”-HMPA0 injection, a mean value being used in subsequent calculations. SPECT acquisition commenced 10 min after 99Tcm-HMPA0 injection. Hydralazine dose was determined according to age, body weight and acetylator status using an escalating schedule (range 25 to 150 mg; 0.37 to 2.86 mg/kg) [34]. Side-effects were ascertained by questionnaire at the end of the second afternoon. Total activity injected as the lipophilic 9YT~mHMPAO species was calculated from total activity injected (measured in all studies) and radiochemical purity of 99Tc”-HMPA0 (measured immediately prior to injection in 14 studies, the mean value of 85 y0 being used at other times ; the remaining 15% consisted predominantly of free pertechnetate which was assumed not to contribute significantly to activity trapped in the tumour or lung). SPECT data was acquired as previously described [ 331. As the reconstruction algorithm supplied with this SPECT system does not permit the use of more than one attenuation coefficient per set of reconstructions, one of two methods (referred to as methods A and B) was employed to enable an appropriate attenuation correction to be made for the level of the thorax containing the tumour. Method A was employed in 16 patients for whom CT scans were available. Attenuation correction factors were derived from CT scans by measurement of the thickness of tissue (whether lung, bone or soft tissue) traversed by representative photon paths originating in tumour or contralateral lung [ 71. From these studies, mean values of attenuation coefficient appropriate to tumours
286 and contralateral lung in upper, mid and lower zones of the chest were also obtained. .Pairs of attenuation coefficients were used when CT scans were not available (method B, four patients) to produce two sets of reconstructions from each set of acquired data. 64 x 64 pixel tomographic images (pixel size 6 mm) were reconstructed in transaxial and sagittal planes using a ramp-Hanning filter with a cut-off frequency of 0.7/cm. Using method A, no attenuation correction was applied at this stage but using method B, attenuation correction was applied using coefficients obtained as described above and a threshold value of 4% to determine patient outline. Transaxial and sagittal reconstructions were reviewed to determine those which intersected through the centre of the tumour mass (Fig. la,b).
On the selected transaxial view, 99Tcm-HMPA0 uptake within the tumour was determined by the superimposition of two regions of interest (ROI) (Fig. lc). The first ROI was of sufficient size to encompass the whole tumour with a minimum amount of adjacent lung whilst a 3 x 3 pixel ROI was used to define the central part of the tumour in which maximum or minimum values of uptake could be identified. A third larger ROI was placed over normal contralateral lung. This was usually of 15 x 10 pixels unless the contralateral hemithorax contained heart or a second tumour when a smaller ROI was used. Where method B was used, tumour and lung ROIs were superimposed on reconstructed images (at the same level) produced using different attenuation coefficients. For each ROI, the mean number of counts per pixel was recorded. In two cases where the mean b
Fig. 1. (a) Carcinoma of the left lower lobe (VF): transaxial SPECT image through centre of tumour (arrowed); (b) sagittal SPECT image showing level of transaxial image in Fig. (a); (c) as (a) with ROIs superimposed; (d) squamous carcinoma of the left upper lobe bronchus (EC) lying close to the hilum (arrowed): transaxial SPECT image.
287 phantom-based corrections were used to obtain tumour : lung concentration ratios (T,,,,). However, this produces over-correction of tumour : lung ratios when tumours lie close to the hilum (e.g. Fig. Id) or at the lung apex and are not totally surrounded by 99Tc”-HMPA0 activity; in four patients (KW, GW, EC, ME) the correction applied reflected the extent to which normal lung covered the tumour surface (one-third to onehalf).
pixel count in the tumour centre was negative as a result of reconstruction, further calculation of central tumour perfusion could not be performed. Where method A was used, the CT-derived attenuation correction factors were then applied. From the attenuation-corrected mean pixel counts, tumour : lung ratios were calculated for both the whole tumour and the tumour centre. Because of the dependence of apparent activity (i.e. counts/pixel) on source size and position,
TABLE
I
Tumour blood flow before and after hydralazine. Hydralazine dose (mg)
Tumour centre
we
post
hydralazine DW RS JT JH PG
25 50 15 50 50
AW FF WF GH ST KW RC GW HH
100 125 75 100 100 125 125 150 100
LC JE FS VF AB CP EC ME
100 50 15 15 100 150 125 150
Mean SEb n
Peripheral resistance
Tumour blood flow (ml/mm per 100 g) Whole tumour post/ pre
pre hydralazine
post
16.4 9.9 18.5 9.2 13.1 21.8 35.7 23.5 29.0 31.4 37.8 7.4 68.0 0.6 44.4 21.8
21.8 9.1 25.1 7.9 15.4 20.5 56.9 19.8 39.3 29.1 49.6 20.0 72.5 2.1 75.9 33.4
1.33 0.92 1.36 0.85 1.17 0.94 1.59 0.84 1.35 0.94 1.31 2.70 1.07 3.72 1.71 1.53
1.00 0.73 0.64 0.94 1.21
23.3 9.0 44.5 24.1 9.7 11.9
1.54 0.85 0.96 2.00 0.88 0.86
0.58 0.94 0.77 0.47 0.89 0.82
28.3 4.4 22
1.38 0.15 22
1.0 3.6 15.3 3.2 zeroa 6.8 30.3 20.2 25.1 19.1 20.7 8.7 59.0 3.2 29.1 13.4
1.6 2.5 16.9 2.8 zero” 5.9 49.1 19.1 35.1 16.2 22.4 18.5 60.9 4.1 53.9 20.4
1.64 0.70 1.10 0.89
12.4 zeroa 18.2 2.8 13.1 18.0
19.9 zeroa 16.6 5.8 12.3 15.4
1.61 0.91 2.11 0.93 0.85
15.1 10.5 46.4 12.4 11.0 13.8
16.2 3.0 20
20.0 3.8 20
1.28 0.10 20
22.6 3.4 22
0.86 1.64 0.95 1.40 0.85 1.08 2.13 1.03 1.46 1.85 1.52
a Negative counts/pixel in tumour ROI as a result of reconstruction, b Standard error of the mean.
post/ pre
post/ pre
0.46 1.15 0.67 0.76 0.48 0.66 0.74 0.27 0.63
288 To calculate tumour blood flow, it was that the contralateral lung represents geneous background whose concentration can be determined from phantom data. 99Tc”-HMPA0 concentration (C,) was from Cback and Tco,,:
assumed a homo(Cback) Tumour obtained
The total activity trapped in the lungs (calculated using predicted lung volumes [ 131) was subtracted from the total lipophilic 99Tc”-HMPA0 activity injected to give the activity (As,,) reaching the systemic circulation. As the fraction of A_ becoming trapped in the tumour (cdrrected for extraction efficiency, E, taken as 80% [32]) is equal to the fraction of cardiac output (Q) perfusing the tumour, tumour blood flow (qJ may be calculated : 100 qt = Q.C’._ A sys *E D where D = tissue density (taken as 1.04 g * cm - 3, [471). The effect of hydralazine on tumour blood flow was investigated by paired-sample t-test and the relationship between tumour blood flow and physiological parameters by linear regression analysis.
68.0) to 28.3 (range 2.1 to 75.9) ml/min per 100 g (p = 0.007; Table I). Physiological responses to hydralazine and their relationship to side-effects are reported elsewhere [34]. An inverse relationship was seen between central and whole tumour blood flow and peripheral resistance (r = 0.50; p = 0.03 and r = 0.66; p = 0.001, respectively; Fig. 2). Mean increases in central and whole tumour blood flow were 28 and 39%, respectively, when the peripheral resistance fell by between 20 and 40%, and 58 and 103 %, respectively, when peripheral resistance fell by more than 40% but mean changes in blood flow through both tumour regions were less than 5 y0 in seven patients (eight tumours) experiencing minimal change in peripheral resistance (20% or less). In contrast there was no correlation between change in central tumour blood flow and blood pressure (r = 0.06; NS). There was a strong correlation between tumour vascular resistance (tumour centre) and peripheral resistance (r = 0.69; p = 0.0007; Fig. 3). The slope of the regression line was 0.80 (95% confidence limits: 0.39-1.22) indicating that vessels supplying tumours are slightly less sensitive 2.5,
Results All 22 patients completed the study. One patient (JE) developed atrial fibrillation on the second day and received a reduced dose of hydralazine. Tumour blood flow was not quantifiable in the same patient because of extravasation of 99T~mHMPAO (without sequelae) and in another (LC) because of loss of SPECT data prior to analysis. Blood flow through central tumour regions rose by a mean of 28% from 16.2 (range 1.0 to 59.3) to 20.0 (range 1.6 to 60.9) ml/min per 100 g (p = 0.03) following hydralazine and in the whole tumour by a mean of 38% from 22.6 (range 0.6 to
! _,
0.0 0.2
0.4 TOTAL
,
,
,
0.6
0.8
1.o
y= 1.961- 0.946x: r = 0.50
PERIPHERAL post/pm
, . I.2
( 1.4
RESISTANCE
hydralazine
Fig. 2. Change in tumour blood flow (tumour change in peripheral resistance.
centre)
vs.
289
0.2, 0.2
I 0.4
I 0.6
y = 0.189+ 0.804x: I = 0.69 . I . 3 0.8 1.0
PERIPHERAL posllpre
I 1.2
1 1.4
RESISTANCE hydralnzine
Fig. 3. Change in tumour vascular resistance (tumour centre) vs. change in peripheral resistance.
to the vasodilating effects of hydralazine than vessels elsewhere. There was no correlation between change in tumour blood flow following hydralazine and pretreatment blood flow or tumour size. Discomfort due to the arms-above-head position necessary for SPECT was reported by 14 patients and discomfort due to the measurement of blood pressure by one patient. Side-effects due to hydralazine were reported by eight patients and are described elsewhere [ 341; the incidence of side-effects was closely related to the degree of physiological disturbance.
Discussion This study demonstrates an increase in tumour blood flow following single dose oral hydralazine in patients with primary lung tumours. The quantitative method incorporated phantom- and CTderived correction factors applied to SPECT data. Although phantom and patient studies were performed using the same acquisition and reconstruction parameters, errors might have arisen
from the use of sources which contained uniformly distributed activity in contrast to the situation in tumours where perfusion appears to vary from periphery to centre. This would result in greater errors in the estimation of blood flow through the whole tumour. Caution also needs to be exercised in the interpretation of blood flow values for the whole tumour as, inevitably, ROIs include some non-tumour tissue. Errors in the estimation of tumour size or in locating tumours on SPECT images were minimised by selecting radiologically well-defined tumours. The contribution of any such errors would generally be diminished when relative changes are considered. Alternative means of attenuation correction include the use of simultaneous transmission and emission scans [3,27] or CT data may be fed directly to the SPECT system and used to provide pixel by pixel attenuation correction [3 11. Techniques to compensate for the effects of scattered photons by additional data acquisition in a lower energy window [ 2 1] or by a deconvolution procedure [49] have been described. The increase in tumour blood flow observed in this study is in contrast to the effects observed in animals in which hydralazine (upto 20 mg/kg i.v.) produced a fall in tumour blood flow [ 1,15,42,44]. In order to compare oral and intravenous doses, oral doses (in man) need to be divided by a factor of between 3 and 6 [ 261 to take account of extensive first-pass hepatic metabolism. Whilst there are no data on the effects of low-dose hydralazine on tumour blood flow in animals, Okunieff et al. [ 291 have recently reported an improvement in tumour 31P magnetic resonance spectra in mice (consistent with an increase in blood flow) following 0.25 mg/kg hydralazine intravenously. These findings, in conjunction with the dose-dependent response to calcium channel blockers [23,24,48], make it increasingly likely that the class of dilator is less important than dose. On this basis, and assuming that sensitivity to vasodilators is not dependent on tumour site, it is probable that all arteriolar dilators in clinically tolerable (i.e. low) doses would produce increases in tumour blood flow.
290 Differences in rates of tumour growth might also explain variation in sensitivity to vasodilators. In slower growing tumours where nutritive demands increase only gradually hypertrophy of arterial vessels may occur and maintain responsiveness to vasodilators, whilst in faster growing tumours (i.e. in transplantable animal tumours though possibly in some human tumours also) adaptive changes cannot keep pace so that supplying vessels continually exist in a state of near-maximal dilation with the result that responsiveness to vasodilators is effectively lost. A technique which increases blood flow to solid tumours may be of therapeutic benefit. Whilst many solid tumours are known for their intrinsic chemo- and radioresistance, the presence of poorly-perfused regions (as demonstrated in carcinoma of the bronchus [33] and soft-tissue sarcomas [37]) may contribute to treatment failure. The use of angiotensin-induced hypertension to increase tumour blood flow and enhance drug delivery has been explored in man [45]. On the other hand, hydralazine increases blood flow to normal tissues so that the toxicity of drugs or radiation might also be enhanced - though not necessarily to the same extent [ 10,401. Furthermore, hydralazine increases hepatic blood flow [35] which might accelerate drug absorption and metabolism or enhance hepatotoxicity, whilst the increase in renal blood flow without significant change in glomerular filtration rate [ 1 l] might affect renal toxicity. In this study, both side-effects and increased tumour blood flow were directly related to the degree of physiological disturbance so that the use of divided doses would be unlikely to provide additional benefit. The potential hazards of extreme physiological disturbance (i.e. stroke and myocardial infarction) are less in younger patients although larger disturbances would not necessarily be better tolerated. In conclusion, single dose oral hydralazine significantly increases blood flow through human lung tumours by active vasodilation of supplying vessels. Both the magnitude of this change and the incidence of side-effects were related to the degree of physiological disturbance.
Acknowledgements The authors wish to express their gratitude to Prof. G. E. Adams and Dr. P. W. Jones for helpful advice in the preparation of this manuscript; to Drs. J. R. Yarnold, N. T. Cooke, P. W. Jones, J. P. Glees, and P. Mitchell-Heggs for permission to study and report on patients under their care; to Ms. S. Chittenden and Mr. R. Crawford for and to Mr. J. Babich preparing 99Tc”-HMPA0 and Mrs. B. Pratt for measuring the radiochemical purity of 99Tc”-HMPA0. This work was supported by the Cancer Research Campaign.
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of y9mTc- (d,l)-hexamethyl-propylenenamine oxime (HM-PAO) in patients with brain tumour studied by single photon emission computed tomography. Eur. J. Nucl. Med. 12: 417-420, 1986. Ludden, T. M., McNay, J. L., Shepherd, A. M.M. and Lin, M. S. Clinical pharmacokinetics of hydralazine. Clin. Pharmacokinet. 7: 185-205, 1982. Macey, D. C. and Marshall, R. Absolute quantitation of radiotracer uptake in the lungs using a gamma camera. J. Nucl. Med. 23: 731-735, 1982. Milne, E. N. C. Vascular supply of primary and metastatic lung tumours in man. Am. J. Roentgenol. 100: 603-608, 1967. Okunieff, P., Kaalinowski, F., Vaupel, P. and Neuringer, L. J. Effects of hydralazine-induced vasodilation on the energy metabolism of murine tumours studied by in vivo 3’P-Nuclear Magnetic Resonance spectroscopy. J. Natl. Cancer Inst. 80: 745-750, 1988. Overgaard, J. and Skinhoj, E. A paradoxical cerebral hemodynamic effect of hydralazine. Stroke 6: 402-404, 1975. Parker, R. P., Hobday, P. A. and Cassell, K. J. The direct use of CT numbers in radiotherapy dosage calculations for inhomogeneous media. Phys. Med. Biol. 24: 802-809, 1979. Reichmann, K., Biersack, H. J., Hartmann, A., Nierhaus, A., Tsuda, Y., Brassel, F., Rommel, T. and Winkler C. HMPAO kinetics in the baboon brain. Nucl. Med. 27: 109-l 10, 1988. Rowell, N. P., McCready, V. R., Tait, D., Flower, M. A., Cronin, B., Adams, G. E. and Horwich, A. Technetium99m HMPAO and SPECT in the assessment of blood Row in human lung tumours. Br. J. Cancer 59: 135-141, 1989. Rowell, N. P. and Clark, K. The effects of oral hydralazine on blood pressure, cardiac output and peripheral resistance with respect to dose, age and acetylator status. Radiother. Oncol., this issue. Shepherd, A. M. M., Irvine, N. A. and Ludden, T. M. Effect of food on blood hydralazine levels and response in hypertension. Clin. Pharmacol. Ther. 36: 14-18, 1984. Shepherd, A. M.M., Irvine, N. A., Ludden, T. M., Lin, M. S. and McNay, J. L. Effect of oral dose size on hydralazine kinetics and vasodepressor response. Clin. Pharmacol. Ther. 36: 595-600, 1984. Sinnett, H. D., Rowell, N. P., McCready, V. R. and Lawrence, R. Demonstration of blood flow patterns in human soft-tissue sarcomas using technetium-99m labelled HM-PAO. Br. J. Surg. 77: 454-457, 1990. Stratford, I. J., Walling, J. M. and Silver, A. R. J. The differential cytotoxicity of RSU 1069: cell survival studies indicating interaction with DNA as a possible mode of action. Br. J. Cancer 53: 339-344, 1986. Stratford, I. J., Godden, J., Howells, P., Embling, P. and Adams, G. E. Manipulation of tumour oxygenation by hydralazine increases the potency of bioreductive radio-
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sensitizers and enhances the effect of melphalan in experimental tumours. In: Radiation Research, Vol. 2, pp. 737-743. Editors: E. M. Fielden et al. Taylor and Francis, London, 1987. Stratford, I. J., Adams, G. E., Godden, J., Howells, N. and Timpson, N. Potentiation of the anti-tumour effect of melphalan by the vasoactive agent, hydralazine. Br. J. Cancer 58: 122-127, 1988. Tait, D., McCready, V. R. and Ott, R. J. HMPAO assessment of human tumour perfusion. Eur. J. Cancer Clin. Oncol. 23: 789-793, 1987. Trotter, M. J., Acker, B. D. and Chaplin, D. J. Histological evidence for nonperfused vasculature in a murine tumour following hydralazine administration. Int. J. Radiat. Oncol. Biol. Phys. 17: 785-789, 1989. Vaupel, P. and Menke, H. Blood flow, vascular resistance and oxygen availability in malignant tumours upon intravenous flunarizine. Adv. Exp. Med. Biol. 215: 393-398, 1987. Vorhees, H. R. and Babbs, C. F. Hydralazine-enhanced selective heating of transmissible venereal tumour implants in dogs. Eur. J. Cancer Clin. Oncol. 18: 1027-1033, 1982.
45 Wakui, A. and Suzuki, M. Cancer chemotherapy in combination with angiotensin-induced hypertension. Jpn. J. Cancer Chemother. 10: 1577-1583, 1983. 46 Webb, S., Flower, M. A., Ott, R. J., Leach, M. A., Fielding, S., Inamdar, R., Lowry, C. and Broderick, M. D. A review of studies in the physics of imaging by single photon emission computed tomography. In: Recent Developments in Medical and Physiological Imaging, pp. 132-146. Editors: R. P. Clark and M. R. Goff. Taylor and Francis, London, 1986. 47 White, D. R. and Fitzgerald, M. Calculated attenuation and energy absorption coefficients for ICRP reference man (1975) organs and tissues. Health Phys. 33: 73-81, 1977. 48 Wood, P. J. and Hirst, D. G. Calcium antagonists as radiation modifiers: site specificity in relation to tumour response. Int. J. Radiat. Oncol. Biol. Phys. 16: 1141-l 144, 1989. 49 Yanch, J. C., Irvine, A. T., Webb, S. and Flower, M. A. Deconvolution of emission tomographic data: a clinical evaluation. Br. J. Radiol. 61: 221-225. 1988.
and Oncology,
18 (1990) 283-292
283
00742
The effects of single dose oral hydralazine on blood flow through human lung tumours N . P . Rowe11 1,4, M. A. Flower3, V. R. McCready2, ‘Academic
B. Cronin* and A. Horwich’
Unit, Department of Radiotherapy, Departments of 2Nuclear Medicine, and 3Physics as Applied to Medicine, Royal
Marsden Hospital, Sutton, Surrey ,942 5PT, U.K., and 4MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD, U.K.
(Received 24 May 1989, revision received 22 January
Key words: Hydralazine;
1990, accepted
Blood flow; Bronchial carcinoma;
15 February
99Tcm-HMPAO;
1990)
SPECT
Summary Hydralazine has been shown to reduce tumour blood flow and to potentiate the cytotoxicity of melphalan and bioreductive agents in mice. In order to determine whether such a strategy might have clinical potential, a study was undertaken to investigate the effects of hydralazine on blood flow through human tumours. Twenty-two patients with carcinoma of the bronchus received a single oral dose of hydralazine in the range 25 to 150 mg (0.37-2.86 mg/kg) according to age and acetylator status. Tumour blood flow was assessed by single photon emission computed tomography (SPECT) performed 10 min following intravenous 99Tc”-HMPA0 on two occasions 2-8 days apart, the second being performed 60 min after hydralazine administration. In 20 evaluable patients, hydralazine caused a 38% increase in blood flow through the whole tumour ( p = 0.007) and a 28 % increase in flow through the tumour centre (p = 0.03) with greater increases occurring in patients sustaining greater falls in peripheral resistance. Tumour vascular resistance fell indicating active vasodilation in arterioles supplying tumours. Side-effects due to hydralazine were reported by eight patients.
Introduction The blood supply of a tumour may be important both in its natural history, influencing metabolism and growth, and in determining response to treatment. Early investigations into the effects of vasoactive agents on tumour blood flow were unAddress for correspondence:
Dr. N. P. Rowell, Department of Radiotherapy and Oncology, University College Hospital, Gower Street, London WClE 6AU, U.K. 0167-8140/90/$03.50
0 1990 Elsevier Science Publishers
dertaken in order to identify drugs which, by increasing tumour blood flow (and possibly also reducing flow through normal tissues), might enhance the effects of radiation. Although factors governing blood flow through tumours are now better understood [ 181 and the effects of vasodilators on tumour blood flow have been extensively reviewed [ 191, full interpretation of reported studies is only possible when physiological parameters (i.e. blood pressure and cardiac output) have been recorded. Studies which report
B.V. (Biomedical
Division)
284 tumour blood flow in terms of fractional distribution of cardiac output (FDCO) are difficult to interpret because of the increase in cardiac output produced by almost all vasodilators. In this study the term “blood flow” refers always to nutritive perfusion; AV shunting is not assessed. As a result of early studies, Cater [ 51 concluded that blood flow through tumours varied with systemic blood pressure but with the notable exception of the vasoactive amines (e.g. noradrenaline and acetylcholine) which appeared to have direct effects on tumour vessels. These direct effects have been demonstrated by others with vasoactive amines [ 16,221 and more recently with calcium channel blockers [23,24,43,48]. There is now abundant evidence that in spite of the histological appearance of scanty smooth muscle associated with tumour vessels [6], vessels may constrict or dilate in response to drugs and that blood pressure is not the sole determinant of tumour blood flow. Studies with the vasodilator hydralazine have variously reported no effect on tumour blood flow (0.5-1.0 mg/kg iv. in anaesthetised rats) [ 1,6], a 50% decrease (10 mg/kg i.p. in anaesthetised mice) [ 151, a 54-90% decrease (0.5 mg/kg i.v. in anaesthetised dogs) [ 1,441 and a 67% decrease (10 mg/kg i.v. in anaesthetised mice) [42]. Although these results appeared to suggest that hydralazine and calcium-channel blockers produced qualitatively different effects on tumour blood flow, the possibility that the two end-results were simply due to differences in dose could not be excluded. Hydralazine has been available as an antihypertensive for over 30 years and dose-response relationships and pharmacokinetics are well understood [26,36]. Oral medication was chosen for this study because of occasional reports of stroke and myocardial infarction following intravenous administration. In man, blood flow patterns have been investiand single photon gated with 99Tcm-HMPA0 emission computed. tomography (SPECT) in brain turnout-s [2,25], lung tumours [33], breast tumours (Rowell et al., unpublished data) and in tumours in a variety of sites [41]. Except for very
low values, tumour : lung ratios in repeat studies performed after an interval of 3-5 days were within k 15% of initial values [33]. r Studies with phantoms have been undertaken to develop a method for correction of SPECT data so that perfusion in a particular region of interest may be expressed quantitatively (Rowell, unpublished data). The difficulties in relating counts/pixel in reconstructed images to absolute activity in tissue are due principally to the effects of scatter and the inadequacy of attenuation correction [ 12,17,20,46]. Discrepancies are greater for smaller and more centrally placed sources. For this study, primary lung tumours were chosen because in SPECT images most were surrounded by a relatively homogeneous background, a situation easier to simulate in phantom studies. In contrast to metastases, primary lung tumours derive almost all of their blood supply from the systemic circulation [28] so that the amount of a radiopharmaceutical retained in a tumour can be expressed as a fraction of that delivered to the systemic circulation (i.e. FDCO). A reduction in tumour blood flow might be turned to therapeutic advantage as hydralazine potentiates the cytotoxicity of melphalan [40], misonidazole [ 141 and the bioreductive agents RSU 1069 [8,14] and SR4233 [4] in mice. Bioreductive drugs are those which undergo selective activation in hypoxic cells by redox cycling [ 381. Administered to mice postirradiation, hydralazine enhances the effectiveness of misonidazole and RSU1069 in transplantable murine tumours [9,39] but not in human tumour xenografts [ 141. In order to assess whether blood flow reductions might be achieved at clinically tolerable doses, we elected to study the effects of single dose oral hydralazine on blood flow through primary lung tumours.
Patients and methods Twenty-two patients with carcinoma of the bronchus (18 men, 4 women; mean age 66.9 years, range 52 to 74) were studied. All patients with a
285 diagnosis of carcinoma of the bronchus under the age of 75 years were eligible provided that on chest radiographs tumours could be clearly defined and there were no adjacent areas of collapse or consolidation. Patients were excluded if there was a history of stroke or myocardial infarction or if there was evidence of uncontrolled hypertension, uncontrolled cardiac failure or raised intracranial pressure [ 301. Patients with atria1 fibrillation or valvular heart disease were excluded because of greater errors in the estimation of cardiac output. The study was approved by the Royal Marsden Hospital Ethics Committee and informed consent was obtained from each patient. Following initial assessment and bronchoscopy by the referring physician, histological diagnosis remained uncertain in three patients despite a confident clinical diagnosis. Of the remainder, fourteen had squamous carcinoma, two small-cell carcinoma, two adenocarcinoma and one a poorly differentiated carcinoma of uncertain type. In two patients, there were two tumour masses on chest radiograph and CT. At the time of the study, 14 patients, were undergoing investigations prior to radiotherapy and three prior to surgery; five with advanced but asymptomatic disease remained under observation. No patient had received prior radiotherapy. Tumour size was assessed from the chest radiograph. Two perpendicular diameters (X,Y) were measured directly from the PA view and demagnified. Where lateral views were not available or were unhelpful, the third dimension (Z) was calculated as the mean of X and Y. Magnification factors of 1.05 for PA views and 1.08 for lateral views were assumed. Tumour volume was calculated as : Tumour volume = T *(X x Y x Z) Equivalent diameter (assuming a spherical configuration) for use in the correction of SPECT data was then calculated using the same formula (mean 5.5 cm; range 2.3 to 9.3 cm). Radial position was measured from CT scans or, when not available, from SPECT images.
The study required out-patient attendance for two afternoons. Both afternoons began with the patient resting on a couch for 30 min. At the end of this 30-min period on the first afternoon, 400 MBq 99Tc”-HMPA0 (Ceretec, Amersham International plc) was administered intravenously and on the second afternoon, hydralazine tablets (Apresoline, Ciba-Geigy) were given by mouth and 400 MBq 99Tc”-HMPA0 administered 60 min thereafter. Blood pressure and cardiac output were measured noninvasively [ 341 during and 5 min after 99Tc”-HMPA0 injection, a mean value being used in subsequent calculations. SPECT acquisition commenced 10 min after 99Tcm-HMPA0 injection. Hydralazine dose was determined according to age, body weight and acetylator status using an escalating schedule (range 25 to 150 mg; 0.37 to 2.86 mg/kg) [34]. Side-effects were ascertained by questionnaire at the end of the second afternoon. Total activity injected as the lipophilic 9YT~mHMPAO species was calculated from total activity injected (measured in all studies) and radiochemical purity of 99Tc”-HMPA0 (measured immediately prior to injection in 14 studies, the mean value of 85 y0 being used at other times ; the remaining 15% consisted predominantly of free pertechnetate which was assumed not to contribute significantly to activity trapped in the tumour or lung). SPECT data was acquired as previously described [ 331. As the reconstruction algorithm supplied with this SPECT system does not permit the use of more than one attenuation coefficient per set of reconstructions, one of two methods (referred to as methods A and B) was employed to enable an appropriate attenuation correction to be made for the level of the thorax containing the tumour. Method A was employed in 16 patients for whom CT scans were available. Attenuation correction factors were derived from CT scans by measurement of the thickness of tissue (whether lung, bone or soft tissue) traversed by representative photon paths originating in tumour or contralateral lung [ 71. From these studies, mean values of attenuation coefficient appropriate to tumours
286 and contralateral lung in upper, mid and lower zones of the chest were also obtained. .Pairs of attenuation coefficients were used when CT scans were not available (method B, four patients) to produce two sets of reconstructions from each set of acquired data. 64 x 64 pixel tomographic images (pixel size 6 mm) were reconstructed in transaxial and sagittal planes using a ramp-Hanning filter with a cut-off frequency of 0.7/cm. Using method A, no attenuation correction was applied at this stage but using method B, attenuation correction was applied using coefficients obtained as described above and a threshold value of 4% to determine patient outline. Transaxial and sagittal reconstructions were reviewed to determine those which intersected through the centre of the tumour mass (Fig. la,b).
On the selected transaxial view, 99Tcm-HMPA0 uptake within the tumour was determined by the superimposition of two regions of interest (ROI) (Fig. lc). The first ROI was of sufficient size to encompass the whole tumour with a minimum amount of adjacent lung whilst a 3 x 3 pixel ROI was used to define the central part of the tumour in which maximum or minimum values of uptake could be identified. A third larger ROI was placed over normal contralateral lung. This was usually of 15 x 10 pixels unless the contralateral hemithorax contained heart or a second tumour when a smaller ROI was used. Where method B was used, tumour and lung ROIs were superimposed on reconstructed images (at the same level) produced using different attenuation coefficients. For each ROI, the mean number of counts per pixel was recorded. In two cases where the mean b
Fig. 1. (a) Carcinoma of the left lower lobe (VF): transaxial SPECT image through centre of tumour (arrowed); (b) sagittal SPECT image showing level of transaxial image in Fig. (a); (c) as (a) with ROIs superimposed; (d) squamous carcinoma of the left upper lobe bronchus (EC) lying close to the hilum (arrowed): transaxial SPECT image.
287 phantom-based corrections were used to obtain tumour : lung concentration ratios (T,,,,). However, this produces over-correction of tumour : lung ratios when tumours lie close to the hilum (e.g. Fig. Id) or at the lung apex and are not totally surrounded by 99Tc”-HMPA0 activity; in four patients (KW, GW, EC, ME) the correction applied reflected the extent to which normal lung covered the tumour surface (one-third to onehalf).
pixel count in the tumour centre was negative as a result of reconstruction, further calculation of central tumour perfusion could not be performed. Where method A was used, the CT-derived attenuation correction factors were then applied. From the attenuation-corrected mean pixel counts, tumour : lung ratios were calculated for both the whole tumour and the tumour centre. Because of the dependence of apparent activity (i.e. counts/pixel) on source size and position,
TABLE
I
Tumour blood flow before and after hydralazine. Hydralazine dose (mg)
Tumour centre
we
post
hydralazine DW RS JT JH PG
25 50 15 50 50
AW FF WF GH ST KW RC GW HH
100 125 75 100 100 125 125 150 100
LC JE FS VF AB CP EC ME
100 50 15 15 100 150 125 150
Mean SEb n
Peripheral resistance
Tumour blood flow (ml/mm per 100 g) Whole tumour post/ pre
pre hydralazine
post
16.4 9.9 18.5 9.2 13.1 21.8 35.7 23.5 29.0 31.4 37.8 7.4 68.0 0.6 44.4 21.8
21.8 9.1 25.1 7.9 15.4 20.5 56.9 19.8 39.3 29.1 49.6 20.0 72.5 2.1 75.9 33.4
1.33 0.92 1.36 0.85 1.17 0.94 1.59 0.84 1.35 0.94 1.31 2.70 1.07 3.72 1.71 1.53
1.00 0.73 0.64 0.94 1.21
23.3 9.0 44.5 24.1 9.7 11.9
1.54 0.85 0.96 2.00 0.88 0.86
0.58 0.94 0.77 0.47 0.89 0.82
28.3 4.4 22
1.38 0.15 22
1.0 3.6 15.3 3.2 zeroa 6.8 30.3 20.2 25.1 19.1 20.7 8.7 59.0 3.2 29.1 13.4
1.6 2.5 16.9 2.8 zero” 5.9 49.1 19.1 35.1 16.2 22.4 18.5 60.9 4.1 53.9 20.4
1.64 0.70 1.10 0.89
12.4 zeroa 18.2 2.8 13.1 18.0
19.9 zeroa 16.6 5.8 12.3 15.4
1.61 0.91 2.11 0.93 0.85
15.1 10.5 46.4 12.4 11.0 13.8
16.2 3.0 20
20.0 3.8 20
1.28 0.10 20
22.6 3.4 22
0.86 1.64 0.95 1.40 0.85 1.08 2.13 1.03 1.46 1.85 1.52
a Negative counts/pixel in tumour ROI as a result of reconstruction, b Standard error of the mean.
post/ pre
post/ pre
0.46 1.15 0.67 0.76 0.48 0.66 0.74 0.27 0.63
288 To calculate tumour blood flow, it was that the contralateral lung represents geneous background whose concentration can be determined from phantom data. 99Tc”-HMPA0 concentration (C,) was from Cback and Tco,,:
assumed a homo(Cback) Tumour obtained
The total activity trapped in the lungs (calculated using predicted lung volumes [ 131) was subtracted from the total lipophilic 99Tc”-HMPA0 activity injected to give the activity (As,,) reaching the systemic circulation. As the fraction of A_ becoming trapped in the tumour (cdrrected for extraction efficiency, E, taken as 80% [32]) is equal to the fraction of cardiac output (Q) perfusing the tumour, tumour blood flow (qJ may be calculated : 100 qt = Q.C’._ A sys *E D where D = tissue density (taken as 1.04 g * cm - 3, [471). The effect of hydralazine on tumour blood flow was investigated by paired-sample t-test and the relationship between tumour blood flow and physiological parameters by linear regression analysis.
68.0) to 28.3 (range 2.1 to 75.9) ml/min per 100 g (p = 0.007; Table I). Physiological responses to hydralazine and their relationship to side-effects are reported elsewhere [34]. An inverse relationship was seen between central and whole tumour blood flow and peripheral resistance (r = 0.50; p = 0.03 and r = 0.66; p = 0.001, respectively; Fig. 2). Mean increases in central and whole tumour blood flow were 28 and 39%, respectively, when the peripheral resistance fell by between 20 and 40%, and 58 and 103 %, respectively, when peripheral resistance fell by more than 40% but mean changes in blood flow through both tumour regions were less than 5 y0 in seven patients (eight tumours) experiencing minimal change in peripheral resistance (20% or less). In contrast there was no correlation between change in central tumour blood flow and blood pressure (r = 0.06; NS). There was a strong correlation between tumour vascular resistance (tumour centre) and peripheral resistance (r = 0.69; p = 0.0007; Fig. 3). The slope of the regression line was 0.80 (95% confidence limits: 0.39-1.22) indicating that vessels supplying tumours are slightly less sensitive 2.5,
Results All 22 patients completed the study. One patient (JE) developed atrial fibrillation on the second day and received a reduced dose of hydralazine. Tumour blood flow was not quantifiable in the same patient because of extravasation of 99T~mHMPAO (without sequelae) and in another (LC) because of loss of SPECT data prior to analysis. Blood flow through central tumour regions rose by a mean of 28% from 16.2 (range 1.0 to 59.3) to 20.0 (range 1.6 to 60.9) ml/min per 100 g (p = 0.03) following hydralazine and in the whole tumour by a mean of 38% from 22.6 (range 0.6 to
! _,
0.0 0.2
0.4 TOTAL
,
,
,
0.6
0.8
1.o
y= 1.961- 0.946x: r = 0.50
PERIPHERAL post/pm
, . I.2
( 1.4
RESISTANCE
hydralazine
Fig. 2. Change in tumour blood flow (tumour change in peripheral resistance.
centre)
vs.
289
0.2, 0.2
I 0.4
I 0.6
y = 0.189+ 0.804x: I = 0.69 . I . 3 0.8 1.0
PERIPHERAL posllpre
I 1.2
1 1.4
RESISTANCE hydralnzine
Fig. 3. Change in tumour vascular resistance (tumour centre) vs. change in peripheral resistance.
to the vasodilating effects of hydralazine than vessels elsewhere. There was no correlation between change in tumour blood flow following hydralazine and pretreatment blood flow or tumour size. Discomfort due to the arms-above-head position necessary for SPECT was reported by 14 patients and discomfort due to the measurement of blood pressure by one patient. Side-effects due to hydralazine were reported by eight patients and are described elsewhere [ 341; the incidence of side-effects was closely related to the degree of physiological disturbance.
Discussion This study demonstrates an increase in tumour blood flow following single dose oral hydralazine in patients with primary lung tumours. The quantitative method incorporated phantom- and CTderived correction factors applied to SPECT data. Although phantom and patient studies were performed using the same acquisition and reconstruction parameters, errors might have arisen
from the use of sources which contained uniformly distributed activity in contrast to the situation in tumours where perfusion appears to vary from periphery to centre. This would result in greater errors in the estimation of blood flow through the whole tumour. Caution also needs to be exercised in the interpretation of blood flow values for the whole tumour as, inevitably, ROIs include some non-tumour tissue. Errors in the estimation of tumour size or in locating tumours on SPECT images were minimised by selecting radiologically well-defined tumours. The contribution of any such errors would generally be diminished when relative changes are considered. Alternative means of attenuation correction include the use of simultaneous transmission and emission scans [3,27] or CT data may be fed directly to the SPECT system and used to provide pixel by pixel attenuation correction [3 11. Techniques to compensate for the effects of scattered photons by additional data acquisition in a lower energy window [ 2 1] or by a deconvolution procedure [49] have been described. The increase in tumour blood flow observed in this study is in contrast to the effects observed in animals in which hydralazine (upto 20 mg/kg i.v.) produced a fall in tumour blood flow [ 1,15,42,44]. In order to compare oral and intravenous doses, oral doses (in man) need to be divided by a factor of between 3 and 6 [ 261 to take account of extensive first-pass hepatic metabolism. Whilst there are no data on the effects of low-dose hydralazine on tumour blood flow in animals, Okunieff et al. [ 291 have recently reported an improvement in tumour 31P magnetic resonance spectra in mice (consistent with an increase in blood flow) following 0.25 mg/kg hydralazine intravenously. These findings, in conjunction with the dose-dependent response to calcium channel blockers [23,24,48], make it increasingly likely that the class of dilator is less important than dose. On this basis, and assuming that sensitivity to vasodilators is not dependent on tumour site, it is probable that all arteriolar dilators in clinically tolerable (i.e. low) doses would produce increases in tumour blood flow.
290 Differences in rates of tumour growth might also explain variation in sensitivity to vasodilators. In slower growing tumours where nutritive demands increase only gradually hypertrophy of arterial vessels may occur and maintain responsiveness to vasodilators, whilst in faster growing tumours (i.e. in transplantable animal tumours though possibly in some human tumours also) adaptive changes cannot keep pace so that supplying vessels continually exist in a state of near-maximal dilation with the result that responsiveness to vasodilators is effectively lost. A technique which increases blood flow to solid tumours may be of therapeutic benefit. Whilst many solid tumours are known for their intrinsic chemo- and radioresistance, the presence of poorly-perfused regions (as demonstrated in carcinoma of the bronchus [33] and soft-tissue sarcomas [37]) may contribute to treatment failure. The use of angiotensin-induced hypertension to increase tumour blood flow and enhance drug delivery has been explored in man [45]. On the other hand, hydralazine increases blood flow to normal tissues so that the toxicity of drugs or radiation might also be enhanced - though not necessarily to the same extent [ 10,401. Furthermore, hydralazine increases hepatic blood flow [35] which might accelerate drug absorption and metabolism or enhance hepatotoxicity, whilst the increase in renal blood flow without significant change in glomerular filtration rate [ 1 l] might affect renal toxicity. In this study, both side-effects and increased tumour blood flow were directly related to the degree of physiological disturbance so that the use of divided doses would be unlikely to provide additional benefit. The potential hazards of extreme physiological disturbance (i.e. stroke and myocardial infarction) are less in younger patients although larger disturbances would not necessarily be better tolerated. In conclusion, single dose oral hydralazine significantly increases blood flow through human lung tumours by active vasodilation of supplying vessels. Both the magnitude of this change and the incidence of side-effects were related to the degree of physiological disturbance.
Acknowledgements The authors wish to express their gratitude to Prof. G. E. Adams and Dr. P. W. Jones for helpful advice in the preparation of this manuscript; to Drs. J. R. Yarnold, N. T. Cooke, P. W. Jones, J. P. Glees, and P. Mitchell-Heggs for permission to study and report on patients under their care; to Ms. S. Chittenden and Mr. R. Crawford for and to Mr. J. Babich preparing 99Tc”-HMPA0 and Mrs. B. Pratt for measuring the radiochemical purity of 99Tc”-HMPA0. This work was supported by the Cancer Research Campaign.
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