John M. Yuhas

Phase I trials of WR-2721 and cis-platinum

WR-2721 is a sulfhydryl compound which in the animal model improves renal tolerance to cis-platinum (DDP) by factors of 1.3 to 1.7. Phase I trials were initiated to establish the toxicity and dose modification factors when WR-2721 was given prior to escalating doses of DDP. Nineteen patients received 27 courses of WR-2721 (450-910 mg/m2) 20 minutes prior to DDP (50-120 mg/m2). Patients were prehydrated, but no mannitol or other diuretics were administered. Mild, transient nephrotoxicity was observed in only 2 of 15 courses of DDP 80-100 mg/m2 when WR-2721 was given prior to DDP. Although 5 of 9 patients treated with WR-2721 prior to 120 mg/m2 of DDP developed transient nephrotoxicity, their serum creatinines returned to normal baseline values within 1 to 2 weeks. Subsequently, the protocol was modified to include mannitol diuresis. Thirty-four courses of WR-2721 (740 mg/m2) prior to DDP 120-150 mg/m2 with mannitol diuresis were administered. Biweekly serum creatinine and monthly creatinine clearances have remained normal in all patients treated with 120 mg/m2 of platinum and WR-2721. Four of 10 patients treated with 150 mg/m2 of cis-platinum experienced transient nephrotoxicity 5-7 days after treatment. Mild ototoxicity was noted in 4 patients following 150 mg/m2 of DDP. WR-2721 does not appear to protect against the antitumor efficacy of DDP, as 57% of all patients achieved objective partial responses, lasting 1+ to 7+ months. Partial responses occurred in 3/4 (75%) of patients with melanoma and 7/10 (70%) patients with cancer of the head and neck. Compared to retrospective series, our data suggest that WR-2721 may provide some protection against platinum-induced nephrotoxicity, but the dose modification factors remain to be established.

Clinical trials of WR-2721 with radiation therapy

The radioprotector with clinical potential, S-2-(3 aminopropylamino)-ethylphosphorothioic acid (WR-2721) is undergoing two Phase I trials. The objectives of these trials are 1) to determine the maximum tolerated dose (MTD) of WR-2721 in a single dose and 2) to determine the highest dose of WR-2721 that can be tolerated daily in the greatest number of fractions per week. A total of 65 patients have been treated. The single maximum tolerated dose has not yet been reached, though 740 mg/m2 is well tolerated. A single dose of 910 mg/m2 has been successfully administered to one patient. The multiple dose MTD is at an early stage with patients currently receiving 170 mg/m2 four times a week. Among the toxicities noted in both trials are hypotension, hypertension, emesis and somnolence. In addition, in the multiple dose trial there have been three patients who have had allergic reactions including one which was life-threatening. Phase II studies are planned and will begin when the maximum tolerated dose is established from each Phase I trial.

Combined use of radioprotective and radiosensitizing drugs in experimental radiotherapy.

The application of both radioprotective and radiosensitizing drugs in clinical radiotherapy is limited by the large drug doses required to obtain significant therapeutic gains. A potential means of circumventing these toxicity problems, while retaining significant therapeutic gains, would be to use small doses of both types of drugs. This possibility has been tested, using the specific drug combination WR-2721 and Ro-07-0582 with the following results: The two drugs are not additive in terms of lethal toxicity in the mouse; WR-2721 does not interfere with the ability of Ro-07-0582 to sensitize tumors in vivo; and Ro-07-0582 does not interfere with the ability of WR-2721 to protect mouse skin. While the therapeutic gains depend on the radiation dose and endpoints considered, the combination proved to be superior to either drug alone in all cases. The success of these preliminary investigations would appear to warrant further study.

PERFLUOROCARBON EMULSIONS IN CANCER THERAPY: PRELIMINARY OBSERVATIONS ON PRESENTLY AVAILABLE FORMULATIONS

Perfluorocarbon (PFC) emulsions, due to their favorable oxygen transporting properties, have been proposed as tumor sensitizers for application in both radiotherapy and chemotherapy. While this application is a very promising one it is by no means simple, and presently available formulations are inadequate. Intravenous administration of these emulsions can produce a severe hemodilution which tends to offset the desired effect; these emulsions can alter the pharmacokinetics of simultaneously administered drugs. Unless these variables are taken into account the risk of false negative and false positive results will be excessive. Of more serious concern are the profound disturbances produced by these emulsions in the reticuloendothelial system. Using two of the more popular PFC, Fluosol-DA and DMA/NONANE, we have shown that daily administration of these emulsions can produce 9-fold increases in liver size and 27-fold increases in spleen size. This problem appears to involve the surfactant used in both emulsions, pluronic F-68. It will be necessary to circumvent this problem before further study of their potential application can proceed.

Phase I Controlled trials of WR-2721 and cyclophosphamide

WR-2721 is an organic thiophosphate compound which in the animal model selectively protects against the hematologic toxicity of cyclophosphamide by factors of 1.5 to 2.0. Controlled Phase I trials of WR-2721 and cyclophosphamide were initiated to determine if WR-2721 protected against cyclophosphamides hematologic toxicity. Fifteen patients received WR-2721 (450-1100 mg/m2) prior to cyclophosphamide (1200-1800 mg/m2) and were subsequently retreated 4 weeks later with the same cyclophosphamide dose alone. With WR-2721 pretreatment, 11/15 (73%) patients had improved WBC counts. The mean WBC increased from 1800/mm3 on cyclophosphamide alone to 2700/mm3 with WR-2721 + cyclophosphamide (p = 0.008). In 11 patients who had nadir differential counts performed, 7 (64%) demonstrated improved nadir granulocyte counts with WR-2721. The mean granulocyte count increased from 765/mm3 on cyclophosphamide to 1274/mm3 with WR-2721 + cyclophosphamide (p = 0.05). In the second trial, 25 patients received the reverse sequence: an initial dose of cyclophosphamide (1200-1800 mg/m2) alone, followed 4 weeks later by WR-2721 (450-1100 mg/m2) prior to the same dose of cyclophosphamide. With WR-2721 pretreatment, 12/25 (48%) patients had improved nadir WBC counts. The mean WBC increased from 1550/mm3 on cyclophosphamide alone to 1850/mm3 with WR-2721 + cyclophosphamide (p = 0.02), while the nadir granulocyte count increased from 449/mm3 to 844/mm3 (p = 0.001). No patient developed microscopic or gross hematuria or inappropriate antidiuretic hormone secretion. One patient developed mild thrombocytopenia. These data suggest that WR-2721 provides significant protection against cyclophosphamide-induced granulocytopenia, but the dose modification factors and degree of clinical benefit remain to be established. The current recommended WR-2721 dose for Phase II trials is 740 mg/m2 administered over 15 minutes.

Phase I clinical trials of WR-2721 with alkylating agent chemotherapy

Abstract WR-2721 is an organic thiophosphate which in the animal model provides selective protection of normal tissues against the toxicity of radiation and alkylating agent chemotherapy. Phase I trials of WR-2721 in man have been initiated to establish the dose modification factors for this agent when used in combination with escalating doses of alkylating agents beyond the level currently tolerated, and to determine whether protection of normal tissues and tumors occurs. Three Phase I trials are in progress. Initially, we used a fixed 450 mg/M2 dose of WR-2721 prior to escalating doses of either cyclophosphamide (CYC) or cis-platinum (PL) as single agents. More recently, the current maximum tolerated dose of WR-2721(740 mg/M2) was administered prior to PL and to nitrogen mustard (HN2). Ten patients received 19 courses of CYC after 450 mg/M2 of WR-2721. At the 1800 mg/M2 LV. dose level of CYC, moderate leukopenia was noted with a median WBC nadir of 1800/mm3 on day 10. Twenty-two courses of PL were administered to 13, patients after 450–740 mg/M2 of WR-2721. Hydration was used, but no mannitol diuresis was employed. Transient nephrotoxicity was noted in five courses at PL doses between 80–120 mg/M2. Mild to moderate leukopenia has been noted with HN2 at 10–16 mg/M2 following WR-2721 (450–750 mg/M2). Objective anti-tumor responses have been observed. These preliminary results suggest that WR-2721 may provide normal tissue protection when administered prior to cyclophosphamide or nitrogen mustard, although the level of protection is not yet of clinical benefit. However, using a 450–740 mg/M2 dose of WR-2721 did not provide protection against platinum-induced nephrotoxicity. WR-2721 does not appear to protect against the anti-tumor activity of alkylating agents (objective tumor responses were noted), nor does there appear to be increased acute toxicity when it is combined with alkylating agent chemotherapy.

Efficacy testing of WR-2721 in Great Britain or everything is black and white at the gray lab

In this issue of the journal, Stewart et ~1.‘~ report their studies on the ability of WR-2721 (S-2-(3-aminopropylamino)ethylphosphorothioic acid) to protect three different murine tumors from ionizing radiation. Most investigators in this area are familiar with the conclusions reached, since they have been presented at four national and international meetings over the past few years,4,5*‘2,‘3 but only in summary form. With publication of the present data, it is now possible to examine the validity of these conclusions, which bear heavily on the proposed use of this type of drug in combination with radiation theraPY. Two major conclusions are reached by these investigators. The first is that “similar protection factors can be achieved in experimental tumors and in normal mouse skin for equal administered doses of WR-2721 and for the same interval between injection and irradiation.” In fact, these investigators have pointed out repeatedly that WR2721 can increase the radiation resistance of one of their tumors by a factor of 2.8,4,5.‘2.‘3,‘5 but the maximum protection they observe in the skin amounts to a 1.6-fold increase.14 Second, and perhaps even more surprising, they compare their skin data with published reports on tumor protection and suggest a “remarkable similarity” to their own data. This latter conclusion was of particular interest, since a number of investigators, including ourselves, had thought they had seen more protection in the skin than in the tumor.2~8~9~“~‘7~‘8~23~24 Although the data are presented in derived form, it is possible to examine them more closely than has been possible in the past, and in doing so we have not come to the same conclusions. Consider first Stewart et ~1’s’~ argument that published reports on tumor protection are consistent with their conclusion of equal protection of skin and solid tumors. This argument is summarized in Figure 5b of their report, and, we must agree that, as plotted, there is little evidence of greater protection of skin than of tumors. The point is, however, that the plot is an invalid one for at least three reasons: inclusion of inappropriate data, inaccurate plotting of other data, and the irrelevance of the comparison itself. First, two of the data points, including the remarkable tumor protection at the top of the plot (Figure 5b) are for leukemias, and these types of tumors have never been a part of the proposal for use of WR-2721 in radiation therapy. At the outset,23 we assumed that the differential protection we were observing was the product of deficient vascularity and/or blood flow, so there was no logical basis for including them in the proposal. Harris and Phillips6 demonstrated protection of the P388 ascitic leukemia by WR-2721 soon thereafter, which only reinforced the exclusion of leukemias from the proposed application. Our present understanding of the problem suggests that leukemias will be well protected by WR2721 for a number of reasons: ascitic tumors will have ready access to intraperitoneally injected WR-2721; in the early stages of growth, such as is the case here, the leukemias are well-oxygenated and this tends to optimize protective drug effectiveness? leukemias apparently lack the membrane restriction to the absorption of WR-2721, which is found in most solid tumors;2’ and ascitic fluid can convert WR-2721 into a radioprotector which is readily absorbed by normal and tumor tissues alike.*’ Since we know that WR-2721 does protect ascitic leukemias and why it does so, inclusion of them in any plot which purports to evaluate the efficacy of WR-2721 in radiation therapy is misleading. Even when we exclude these data points for the leukemias from Figure 5b,” the remaining plot does not provide strong evidence for greater protection of the skin than of the tumors. This raises the second problem with this comparison: some of the data have been plotted incorrectly. Lowy and Baker’ reported that WR-2721 increased the resistance of the KHT sarcoma by a factor of 1.2, not 1.4 as plotted in Figure 5b. In our initial studies on a mammary carcinoma,23 we reported a tumor protection factor of 1.14 (cure) or 1. I5 (time to produce takes), not a factor of 1.3 as plotted in Figure 5b. Last, in our studies on a murine lung adenoma,” we observed no

Variation in normal tissue responsiveness to WR-2721

The radioprotection afforded normal tissues by WR-2721 or S-2-[3-aminopropylamino]ethylphosphorothioic acid varies widely, with some of the most responsive tissues showing low levels of absorbed drug and vice versa. While the oxygen tension of the respective tissues may be contributing to this variation, it can be shown that the drug is not homogeneously distributed within each tissue, and that its location within the sub-cellular compartments can vary widely. Recognition of this variability, when combined with the newly developed HPLC assays for drug quality, should provide new insights into the sources of variation in normal tissue responsiveness to these agents.

Response of human neuroblastoma and melanoma multicellular tumor spheroids (MTS) to single dose irradiation

The growth characteristics of 6 human cell line derived multicellular tumor spheroids (MTS) were studied. Melanoma MTS (C32, HML-A, HML-B) were slow growing with baseline growth rates of 13.9 to 27.3 microns diameter/day. Neuroblastoma MTS (Lan-1, NB-100, NB-134) grew rapidly, with baseline growth rates of 32.1 to 40.3 microns diameter/day, that is, 1.2 to 2.9 times as fast as the melanomas. Delay constants were calculated for all six lines. The neuroblastomas were more sensitive to radiation than melanomas, as reflected in a greater value for the radiation-induced growth delay constant. One neuroblastoma line, Lan-1, was highly radioresponsive; that is, after a subcurative dose of radiation, the MTS diameter decreased beyond the original diameter, which was followed by recovery and regrowth. Irrespective of these initial changes in diameter, growth delay sensitivity (value of delay constant) was the same for Lan-1 and NB-100, an MTS line that did not show the responsive pattern.

Late effects of unilateral radiation on the mouse kidney.

A system is described for quantitative assessment of renal radiation damage in intact animals. Mice received 1000, 2000, 3000, or 6000 rad of single-dose x irradiation to the right kidney. Animals were sacrificed 6 months after irradiation, the ratio of left to right renal weight and the ratio of left to right renal weight showed reasonably good correlation with radiation dose, the latter comparison appearing to be more sensitive, due to the amplification of effect resulting from left renal hypertrophy and right renal atrophy. Mice receiving 1000 rad showed no significant histologic abnormalities, but evidenced definite tubular epithelial atrophic changes after doses of 2000 rad or greater.