Joseph F. Gross

A transparent access chamber for the rat dorsal skin fold.

Abstract The design and surgical implantation procedure of a new aluminum chamber with a transparent window for the rat dorsal skin fold are described. The chamber combines strength, low weight, and high optical transparency, and provides access to the subcutaneous tissue. The latter permits transplantation of different tissue, application of chemical substances, or micromanipulation. In the present work, a mammary adenocarcinoma was transplanted into the subcutaneous tissue within the chamber for observation of tumor growth and microcirculation.

Analysis of the effects of oxygen supply and demand on hypoxic fraction in tumors.

The extent of hypoxic regions in a tumor tissue depends on the arrangement, blood flow rate and blood oxygen content of microvessels, and on the tissues oxygen consumption rate. Here, the effects of blood flow rate, blood oxygen content and oxygen consumption on hypoxic fraction are simulated theoretically, for a region whose microvascular geometry was derived from observations of a transplanted mammary andenocarcinoma (R3230AC) in a rat dorsal skin flap preparation. In the control state, arterial PO2 is 100 mmHg, consumption rate is 2.4 cm3 O2/100 g/min, and hypoxic fraction (tissue with PO2 < 3 mmHg) is 30%. Hypoxia is abolished by a reduction in consumption rate of at least 30%, relative to control, or an increase in flow rate by a factor of 4 or more, or an increase in arterial PO2 by a factor of 11 or more. These results suggest that reducing oxygen consumption rate may be more effective than elevating blood flow or oxygen content as a method to reduce tumor hypoxia.

Analysis of oxygen transport to tumor tissue by microvascular networks

We present theoretical simulations of oxygen delivery to tumor tissues by networks of microvessels, based on in vivo observations of vascular geometry and blood flow in the tumor microcirculation. The aim of these studies is to investigate the impact of vascular geometry on the occurrence of tissue hypoxia. The observations were made in the tissue (thickness 200 microns) contained between two glass plates in a dorsal skin flap preparation in the rat. Mammary adenocarcinomas (R3230 AC) were introduced and allowed to grow, and networks of microvessels in the tumors were mapped, providing data on length, geometric orientation, diameter and blood velocity in each segment. Based on these data, simulations were made of a 1 mm x 1 mm region containing five unbranched vascular segments and a 0.25 mm x 0.35 mm region containing 22 segments. Generally, vessels were assumed to lie in the plane midway between the glass plates, at 100 microns depth. Flow rates in the vessels were based on measured velocities and diameters. The assumed rate of oxygen consumption in the tissue was varied over a range of values. Using a Greens function method, partial pressure of oxygen (PO2) was computed at each point in the tissue region. As oxygen consumption is increased, tissue PO2 falls, with hypoxia first appearing at points relatively distant from the nearest blood vessel. The width of the well-oxygenated region is comparable to that predicted by simpler analyses. Cumulative frequency distributions of tissue PO2 were compared with predictions of a Krogh-type model with the same vascular densities, and it was found that the latter approach, which assumes a uniform spacing of vessels, may underestimate the extent of the hypoxic tissue. Our estimates of the maximum consumption rate that can be sustained without tissue hypoxia were substantially lower than those obtained from the Krogh-type model. We conclude that the heterogeneous structure of tumor microcirculation can have a substantial effect on the occurrence of hypoxic micro-regions.

Morphologic and hemodynamic comparison of tumor and healing normal tissue microvasculature

The purpose of this study was to compare microvascular morphometric and hemodynamic characteristics of a tumor and granulating normal tissue to develop quantitative data that could be used to predict microvascular characteristics which would be most likely associated with hypoxia. The dorsal flap window chamber of the Fisher 344 rat was used to visualize the microvasculature of 10 granulating and 12 tumor (R3230 AC adenocarcinoma) tissues at 2 weeks following surgical implantation of the chamber. Morphometric measurements were made from photomontages and video techniques were used to assess red cell velocities in individual vessels. The percent vascular volume of both tissues was close to 20%, but significant differences were noted in other morphometric and hemodynamic measurements. Individual vessel dimensions (length and diameter) in tumors averaged twice as large as those in granulating tissues. Furthermore, red cell velocities were twice as high in tumors as in granulating tissues. In addition to these large differences in average values, there was significant heterogeneity in tumor microvascular morphometry, indicating spatial nonuniformity compared with the granulating tissue. Approximations of vessel spacing, indicated an average of 257 and 118 microns in tumors and granulating tissues, respectively. Vessel densities were four times greater in granulating tissues than in tumor tissues. These results indicated that intervessel distances were more likely to result in hypoxia in tumors, especially considering the wide variability in that tissue. Analysis of flow branching patterns showed that vascular shunts occurred frequently in vessels ranging from 10 to 90 microns in diameter. The results of this study indicate, in this tumor model, that conditions such as low vascular density, vascular shunts, excessive vascular length and/or low red cell velocity exist to a greater extent than the granulating tissue control. These conditions are likely to be conductive to the development of hypoxia.

Estimation of tissue-to-plasma partition coefficients used in physiological pharmacokinetic models

An important parameter in the development of pharmacokinetic models is the ratio of tissue drug concentration to the concentration of the drug in the arterial plasma or the effluent plasma. The relationship between these two tissue/plasma ratios is derived analytically for different routes of drug administration. The two are equal only in compartments with no elimination when the drug is infused at constant rate. For other routes of administration, the two ratios are identical in all compartments only when there is no elimination process. The tissue/plasma concentration ratios for infusion equilibrium are not equal to the corresponding values for the postdistribution phase after an intravenous bolus injection. When the plasma concentration for infusion and injection are the same, more drug will appear in the lung during infusion steady state than during the postdistribution equilibrium. The reverse is true for the other organs. The importance of properly defining the tissue/plasma ratio and its implication for pharmacokinetic modeling are discussed. The results may have important therapeutic implications for the availability of drugs using different routes of administration.

Perivascular Oxygen Tensions in a Transplantable Mammary Tumor Growing in a Dorsal Flap Window Chamber

Fischer 344 rats with R3230 Ac mammary carcinomas implanted in dorsal flap window chambers served as a model to obtain measurements of perivascular and stromal oxygen tension in normal and tumor tissues using Whalen recessed-tip microelectrodes (3- to 6-microns tip). Perivascular measurements were made adjacent to vessels with continuous blood flow. Thus the measurements and models provided are reflective of conditions leading to chronic hypoxia. Perivascular oxygen tensions averaged 72 +/- 13 mmHg in normal tissue vessels adjacent to tumor, 26 +/- 5 mmHg in tumor periphery, and 12 +/- 3 mmHg in tumor central vessels. There was a significant trend toward lower perivascular oxygen tensions in the tumor center (Kruskal-Wallis test, P = 0.002). A similar tendency was seen with a limited number of stromal measurements. Krogh cylinder models, which incorporate these data for perivascular oxygen tension, along with morphometric data obtained from the same tumor model suggest that hypoxic regions will exist between tumor vessels in the tumor center unless O2 consumption rates are well below 0.6 ml/100 g/min. The low perivascular measurements observed near the tumor center combined with the theoretical considerations suggest, for this model at least, that tissue oxygenation may best be improved by increasing red cell velocity and input pO2 and reducing oxygen consumption. The low perivascular oxygen tensions observed near the center also suggest that conditions conducive to increased red cell rigidity exist, that drugs which can decrease red cell rigidity could improve tumor blood flow and oxygenation, and that the endothelium of those vessels may be susceptible to hypoxia-reoxygenation injury.

Kinetics of intravenous melphalan

We have studied the disposition and elimination of melphalan after intravenous administration in 9 patients with cancer. High‐pressure liquid chromatography and 14C‐melphalan were used to assay drug concentration in plasma and urine. Composite plasma t½β was 7.7 ± 3.3 and t½β was 108 ± 20.8 min for 8 of the patients. The mean 24‐hr urinary excretion of melphalan was 13.0 ± 5.4% of the administered dose. In 2 patients, 80% to 100% of the measured 14C counts in plasma and urine samples at each study interval, up to 24 hr after drug administration, could be accounted for by the sum of parent compound, monohydroxy and dihydroxy products, and methanol nonextractable radioactivity (i.e., protein‐bound activity). These data and evidence of rapid disappearance from plasma at 37° in vitro suggest that spontaneous degradation, and not enzymatic metabolism, is the major determinant of the t½β of melphalan in vivo.

Theoretical Simulation of Oxygen Transport to Tumors by Three-Dimensional Networks of Microvessels

Hypoxic cancer cells are resistant to irradiation and to some types of chemotherapy. When these methods are used to treat tumors, presence of small hypoxic regions can compromise the outcome. Therefore, it is important to understand the factors determining the partial pressure of oxygen (pO2) in tumors at the microscopic level. These factors include the oxygen supply to the tumor, which depends on the flow rate and oxygen content of blood and the arrangement of microvessels within the tissue, and the rate of oxygen consumption by the tissue (Vaupel, 1979).

Bleomycin pharmacokinetics in man: II. Intracavitary administration

Disposition of bleomycin was studied in plasma and urine (14 patients) and ascites fluid (2 patients) after intraperitoneal (IP) and intrapleural (IPl) administration, by radioimmunoassay. Peak plasma bleomycin concentrations after 60 U/m2 in 12 patients ranged between 0.4 and 5.0 mU/ml. For those patients with creatinine clearances greater than 50 ml/min the composite terminal phase bleomycin plasma half-lives (±SD) for three ‘IPl’ and six ‘IP’ patients were 3.4±0.3 and 5.3±0.4 h, respectively. The composite IP plasma half-life was significantly longer than the IPl hal-life (P<0.001) and previously reported IV half-life (t1/2=4.0 ±0.6 h) (P<0.01). In patients with normal renal function, bleomycin excretion during the first 24 h was in most cases lower following intracavitary (IC) than following IV administration (21.7%±8.6% vs. 44.8%±12.6%, respectively) (P<0.005). Comparison of bleomycin plasma concentration time products normalized for dose and half-life for IV and IC administration allowed an estimate that about 45% of the IC bleomycin dosage is absorbed into the systemic circulation. When calculating the total systemic exposure to bleomycin for a patient we suggest using the sum of the IV dose and one-half of the IC dose.

Bleomycin pharmacokinetics in man. I. Intravenous administration.

SummaryDisposition of bleomycin was studied in plasma and urine (14 patients) and ascites fluid (2 patients) after intraperitoneal (IP) and intrapleural (IPl) administration, by radioimmunoassay. Peak plasma bleomycin concentrations after 60 U/m2 in 12 patients ranged between 0.4 and 5.0 mU/ml. For those patients with creatinine clearances greater than 50 ml/min the composite terminal phase bleomycin plasma half-lives (±SD) for three ‘IPl’ and six ‘IP’ patients were 3.4±0.3 and 5.3±0.4 h, respectively. The composite IP plasma half-life was significantly longer than the IPl hal-life (P<0.001) and previously reported IV half-life (t1/2=4.0 ±0.6 h) (P<0.01). In patients with normal renal function, bleomycin excretion during the first 24 h was in most cases lower following intracavitary (IC) than following IV administration (21.7%±8.6% vs. 44.8%±12.6%, respectively) (P<0.005). Comparison of bleomycin plasma concentration time products normalized for dose and half-life for IV and IC administration allowed an estimate that about 45% of the IC bleomycin dosage is absorbed into the systemic circulation. When calculating the total systemic exposure to bleomycin for a patient we suggest using the sum of the IV dose and one-half of the IC dose.SummaryBleomycin plasma decay kinetics and urinary excretion were studied in nine patients after IV bolus injections of 13.7 to 19.9 U/M2. Radio-immunoassay was used to measure bleomycin in plasma and urine samples. The resulting plasma concentration versus time data for each patient and the combined data obtained from all patients were fitted to a multiexponential equation using a nonlinear regression computer program. Pharmacokinetic parameters derived from the mean of all individual patient parameters and the composite of all plasma decay data were similar. Bleomycin initial and terminal plasma half-lives and volume of distribution for all plasma decay data from eight patients with normal serum creatinies were 24.4±4.0 min, 237.5±8.5 min, and 17.3±1.5 L/M2, respectively. Mean 24-h urinary excretion accounted for 44.8±12.6% of the dose in seven patients who had normal serum creatinine values and complete urine collections. The total body clearance and renal clearance in these seven patients averaged 50.5±4.1 ml/min/M2 and 23.0±1.9 ml/min/M2, respectively. One patient with a serum creatinine of 1.5 mg% (normal 0.7 to 1.3 mg%) who was given 15.6 U/M2 had a terminal plasma halflife of 624 min, a volume of distribution of 36.3 L/M2, and 24-h urinary excretion of 11.6% of the dose. We conclude that bleomycin after intravenous bolus injection has a relatively short terminal phase plasma halflife and relatively large urinary elimination.