Dennis B. Leeper

pH DISTRIBUTION IN HUMAN TUMORS

The effectiveness of hyperthermia in tumor therapy may depend on a lower extracellular pH of tumor compared to that of normal tissue. A technique for measuring extracellular pH in human tumors has been devised to test the usefulness of this parameter as prognostic indicator of tumor hyperthermia response. In a preliminary study 50 of 53 pH readings from 14 human tumors (both heated and unheated) were below normal physiological pH. Tumor pH values ranged between 5.55-7.69 (average for unheated tumors 6.81 +/- 0.09, SEM, only one determination was above 7.40). Although there was considerable heterogeneity of pH within tumors, the accuracy and drift of the 21 gauge needle electrode were not a problem. Fifteen minutes were required for pH stabilization after insertion of an 18 gauge open-ended catheter, and less than 5 min for equilibration after electrode insertion into the catheter. A saline wheal was used for anesthesia to preclude modification of pH by anesthetics. Central portions of tumors were no more acidic than peripheral regions, but large tumors tended to be more acidic than small tumors. The pH of several tumors of various sizes and histologies was also determined immediately before subsequent treatment sessions. These measurements were made by reinsertion of catheters in approximately the same locations at each session. The trend appeared to be that pH increased with the number of treatment sessions. Measurements of pH were made in four patients immediately prior to and at the termination of a heating session (same locations since catheter remained in place during heating sessions). Three of the four tumors showed increased pH readings of 0.25-0.54 units during heating. However, none of the four tumors achieved temperatures exceeding 42 degrees C. The pH measurement technique developed provides a safe and relatively easy method for determining extracellular pH in human tumors. There appears to be a correlation of pH values with tumor size, treatment session, and possibly blood flow.

Response of human tumor blood flow to local hyperthermia

The effect of heat on blood flow in human tumors was studied as a function of time during 1 hour of local hyperthermia induced by 915 MHz microwaves. Blood flow was determined from the rate of thermal clearance by use of the bio-heat transfer equation. The rate of thermal clearance was measured at intervals of approximately 10 minutes throughout the treatment session by turning off the microwave power for 50 seconds. Tumor blood flow increased by amounts varying from 15 to 250% during the first 20-50 minutes of heating at 41-45 degrees C, after which it remained relatively constant during the remainder of the treatment session. The sharp reduction in blood flow or vascular stasis reported in most transplantable rodent tumors after comparable heating was not observed in human tumors. The maximum blood flow observed in heated human tumors ranged from 10-40 ml/min/100 gm. The systematic error due to thermal conduction was estimated to be equivalent to a blood flow of less than 3 ml/min/100 gm.

Modification of human tumor pH by elevation of blood glucose

Nine patients with metastatic or recurrent superficial tumors of varying size and histology were administered 100 g oral glucose to investigate whether hyperglycemia can selectively lower tumor pH. pH was measured by a 21 ga modified glass needle electrode inserted through an 18 ga open-ended Angiocath. Serum glucose was monitored every 7.5 min by finger stick and a blood glucose analyzer. Tumor pH was measured over 50-80 min concomitantly with determination of blood glucose. In five nondiabetic patients (eight measurement points) tumor pH decreased 0.05-0.5 units from a pre-glucose range of 6.8-7.4 (7.14 +/- 0.08) to 6.4-7.3 (6.90 +/- 0.10) as blood glucose increased from a baseline of 80-120 mg/dl to 165-215 mg/dl. There was considerable heterogeneity from patient to patient regardless whether blood glucose increased to a peak at 40-60 min post-ingestion and then decreased, or whether it remained elevated up to the end of the 80 min observation period. In general, tumor pH decreased as blood glucose increased and then continued to fall throughout the period of observation. In one patient, tumor pH did not change although blood glucose increased to 175 mg/dl. Normal tissue pH was 7.36 +/- 0.02 when determined on four occasions in three patients (subcutaneous and intramuscular sites), and was unaffected by glucose administration. As a further control for tumor tissue and pH probe stability, pH probes in two patients were left in place for 30 min before glucose ingestion. The tumor pH was stable for the entire interval. Interestingly, three patients had an abnormal glucose response: two of those patients (one patient on two separate occasions) had an increase in blood glucose to 230-260 mg/dl in 40-60 min and pH actually increased 0.1-0.3 units. The third patient had a transient increase in blood glucose to 290 mg/dl along with a corresponding increase and subsequent decrease in tumor pH. In summary, whether glucose was given pre- or post-hyperthermia, independently of position in the tumor, and independently of whether pH increased or decreased, the slope of the curve of pH = f(time) was similar in a given patient tumor on all measurement occasions. These preliminary results suggest that hyperglycemia may be useful in non-diabetic patients, and perhaps in diabetic patients given insulin, to selectively reduce tumor pH and sensitize tumors to hyperthermia.

Interaction of sublethal and potentially lethal 45°-hyperthermia and radiation damage at 0, 20, 37 or 40°C

Abstract The interaction of hyperthermia damage and radiation damage has most often been studied under conditions which favoured repair of sublethal damage. In the present study the interaction of 45°C -hyperthermia damage and radiation damage on the survival of Chinese hamster ovary (CHO) cells was determined at temperatures of 0–4°, 20° and 40°C . Subphysiological temperatures inhibited to various degrees both the repair of sublethal radiation and sublethal 45°C -hyperthermia damage while incubation at 40°C apparently led to the conversion of sublethal 45°C -hyperthermia lesions to lethal lesions. Potentially lethal hyperthermia damage (H-PLD) was repaired at 0–4° and 20°C ; whereas the repair of potentially lethal radiation damage (X-PLD) occurred at 20°C , but not at 0–4° or 40°C . The interaction of 45°C -hyperthermia and radiation damage at temperatures of 0–4°, 20° and 40°C represented a super-position of the separate effects of the two treatment modalities separately combined with incubation at 0–4°, 20° or 40°C . This suggests that ionizing radiation and hyperthermia are affecting distinct targets and that H-PLD and X-PLD do not interact.

Effect of intestinal hyperthermia in the Chinese hamster

If hyperthermia is to become a useful cancer therapeutic modality, normal tissue response must be thoroughly understood. The hyperthermia response of Chinese hamster intestine was studied by immersion of the exteriorized small intestine in heated tissue culture medium. After heating, the small intestine was reinserted, the incision closed, and animals observed until death. Animals exposed to 42.5 degrees, 43.5 degrees, or 44.5 degrees C intestinal hyperthermia exhibited LD50/7 values (including 95% intervals) of 56 min (52.9-59.3), 29 min (26.4-31.8), or 14 min (13.2-14.6), respectively. An Arrhenius plot of LD50/7 vs 1/T degree K exhibited an inactivation energy of 139 kcal/mole, which corresponds well with values generally reported for cellular inactivation. Hamster intestine conditioned with a sublethal exposure of 8 min at 44.5 degrees C developed thermotolerance to subsequent 44.5 degrees C hyperthermia. Thermotolerance induction was maximal by 24 hr; the LD50/7 for the second dose of hyperthermia increased from 6 min at 44.5 degrees C at zero time to 21 min at 44.5 degrees C after a treatment interval of 24 hr (thermotolerance ratio of 3.5). The LD50/7 subsequently decreased from 21 min to 12 min at 44.5 degrees C (the control value) by 96 hr. The hyperthermia response of this tissue was predicated by previous results from the Chinese hamster ovary (CHO) fibroblast cell line in tissue culture, and is also similar to several mouse normal tissues.

The effect of lucanthone (Miracil D) on sublethal radiation damage in chinese hamster cells

Abstract The effect of lucanthone (Miracil D, Nilodin) on the accumulation and repair of sublethal radiation damage was evaluated in Chinese hamster ovary (CHO) cells. Lucanthone is a thiaxanthenone which reversibly inhibits DNA and RNA synthesis by intercalating with DNA and exhibits a concentration-time dependent toxicity resulting in exponential survival curves ( D o ≌ 5 hr for 5 μg/ml, D O ≌ 1.2 hr for 50 μg/ml). When it is present for sufficient time, lucanthone reduces the cells capacity to accumulate and repair sublethal radiation damage. For instance, 5 μg/ml (e.g. a clinically attainable concentration) for 10 hr reduced the D q of the radiation dose-cell survival curve from 200 rad to approximately 40 rad. The effect of lucanthone was manifest whether it was present before or after irradiation. The slope of the radiation dose-cell survival curve was not affected by the presence of lucanthone. Cells recovered the full potential to accumulate sublethal radiation damage very shortly after removal of the drug. Lucanthone (5 μg/ml) present between two fractions of 450 rad reduced the recovery ratio (corrected for lucanthone toxicity) from approximately 2.5 to 1.4 at a fractionation interval of 2 hr, but by 4 hr, the recovery ratio returned to 1.0. When lucanthone was removed immediately before two-dose fractionation, the recovery ratio was similar to the control. Lucanthone also prevented the return of the shoulder if it was present between challenge dose of radiation and an ensuing survival curve determination. Lucanthone (5 μg/ml) reduced the rate of DNA synthesis per cell to approximately 70% of control over a 9 hr period, and raised the 3 H-TdR labeling index from 60 to 70% throughout a 9 hr exposure period. Purely parasynchronization phenomena are unlikely to explain the modifying effects of lucanthone on cellular radiation lethality.

The effect of adriamycin and radiation on G2 cell survival

Abstract The effect of Adriamycin (ADR) and irradiation on the ultimate fate of Chinese hamster ovary cells treated in G 2 , phase was investigated utilizing the mitotic selection procedure for cell cycle analysis coupled with a plating efficiency assay. As cells were selected in mitosis, they were inoculated at appropriate numbers and incubated for analysis of colony-forming ability. The parameters obtained, expressed as a function of time after initiation of treatment, were the number of mitotic cells selected per shake (the mitotic yield), the plating efficiency (surviving fraction), and the number of surviving cells per shake (the product of the first two parameters). Cells treated with a 20 min pulse of 1.0 μ/ml ADR exhibited a transient division delay with a decrease in plating efficiency at the nadir of the mitotic yield curve. Cells treated with 150 rad of X-ray displayed a similar response except that surviving fraction decreased immediately after treatment. As cells recovered from the radiation-induced division delay, the surviving fraction increased to a plateau level close to that of the control. When cells were treated with both ADR and X-ray, the result was an increase in the duration of the division delay, a decreased surviving fraction, and consequently, a decreased number of surviving cells per shake. These results suggest the possibility of predicting optimum schedules for sequential treatment with these two agents based on a knowledge of the perturbatory effects on cell cycle progression for those cells most likely to retain clonogenic ability.

Influence of limb restraint on the thermal response of bone marrow CFU-GM heated in situ.

The method used to restrain anaesthetized (sodium pentobarbital) mice for in situ heating of tibial marrow affects the survival response of CFU-GM. Three methods of limb restraint, in addition to ischaemia induced by tourniquet, were examined for their relative effect on the thermal response of CFU-GM. The three methods of restraint were to secure only the toes with suture material to a submersion post in the water bath, to tape the foot, or to tape the leg. Temperatures in the lumen of the tibia were measured with a 100 micron (tip diameter) microthermocouple during representative experimental conditions. After heating in situ, bone marrow was extruded and CFU-GM cultured in standard soft agar conditions in lung-conditioned medium. The most restrictive restraining method, i.e. taping the leg, produced the greatest thermal response among the three restraining methods examined. The D0 (+/- 95% CI) of the 42 degrees C survival curve for CFU-GM was 22 +/- 4, 46 +/- 8, or 94 +/- 53 min for restraint of leg, foot, or toes, respectively. Survival reached a plateau by 100 min of heating indicative of the development of thermotolerance. The D0 of the 44 degrees C survival curve was 3 +/- 1, 6 +/- 2 and 16 +/- 6 min for restraint of leg, foot, or toes respectively. Ischaemia produced the most pronounced effect on the thermal response of tibial CFU-GM with D0 values of 2 +/- 1 or 3.6 +/- 1.5 min after exposure to 44 degrees C or 42 degrees C, respectively. The method of limb restraint affects the thermal sensitivity of CFU-GM most probably by blood flow obstruction and resultant pH decrease. Thus, precautions must be taken to ensure that limb restriction does not introduce artifacts in the hyperthermia response of normal tissues or tumours during heating in situ.

The effect of lucanthone on sublethal radiation damage, in vivo

The capacity of the Chinese hamster jejunal crypt cell to accumulate and repair sublethal radiation damage was determined by analyzing the return of the shoulder of the radiation dose-crypt microcolony survival curve (Dr) after a priming dose of 1250 rad. The control split dose crypt cell survival curve exhibited a D0, Dr and n of 179 +/- 3 rad, 261 +/- 3 rad and 4.3 respectively; repair of sublethal radiation damage was completed by two hours post-irradiation. The effect of lucanthone (an antischistosomal DNA intercalating agent) on the crypt cells capacity to accumulate and repair sublethal radiation damage was determined by injecting the drug (100 mg/kg, i.p.) at intervals before irradiation with a priming dose of 1250 rad, followed two hours later by graded doses. Injection coincident with the priming dose of radiation resulted in a 22 rad reduction of the Dr (compared to control Dr). Injection eight hours before the priming dose almost completely inhibited the accumulation and repair of sublethal radiation damage so that the resultant Dr two hours later was only 29 rad (a 232 rad reduction). At no time was the D0 of the crypt cell survival curve affected by lucanthone. These data confirm previous results from whole crypt analysis and LD50/7 analysis that non-toxic concentrations of lucanthone reversibly inhibit the accumulation and repair of sublethal radiation damage in a time-dependent manner with complete inhibition approximately eight hours post-injection. This drug is useful for the study of sublethal radiation damage in vivo and may be beneficial in radiation therapy of cancer when it is desirable to inhibit the repair of sublethal radiation damage.

Lucanthone modification of cyclophosphamide toxicity in the Chinese hamster.

The interaction of lucanthone and cyclophosphamide (CYC) was investigated in the Chinese hamster in terms of the LD50/7 and LD50/30. These values may be indicative of gastrointestinal stem cell depletion and bone marrow stem cell depletion, respectively. When a nonlethal dose of 100 mg/kg lucanthone preceded CYC injection, the LD50/7 for CYC reached its minimum value of 470 mg/kg at a treatment interval of 10 hours. Lucanthone administered simultaneously with CYC had no effect on the control LD50/7 of 750 mg/kg, and by 48 hours after lucanthone administration the LD50/7 had returned to the control value. When CYC administration preceded that of lucanthone, the LD50/7 reached a minimum of value of 610 mg/kg at an interval of 5 hours; however, for the entire sequence it was approximately 640 mg/kg over all intervals up to 48 hours. The LD50/30 for CYC was only slightly reduced by the presence of lucanthone, indicating that bone marrow sensitivity to CYC was only marginally affected by lucanthone. These data indicate that lucanthone may interact with CYC damage in much the same way as it interacts with radiation damage, viz, by reducing cellular capacity to accumulate and repair sublethal damage.