Isaac Djerassi

Phase I study of high doses of methotrexate with citrovorum factor in patients with lung cancer

A Phase 1 study of high‐dose “pulse” methotrexate infusions combined with citrovorum factor “rescue” in advanced lung cancer indicated consistent tumor response in eight patients. A dose schedule allowing for repeated treatments, and thus suitable for prolonged management, was developed. Three patients treated accordingly for 8 to 10 months continue to show regression of their original lesions without evidence of new metastases. These observations are consistent with previously proposed concepts for use of citrovorum “rescue” after administration of high‐dose “pulses” of folic acid antagonists.

Methotrexate and citrovorum factor rescue in the management of childhood lymphosarcoma and reticulum cell sarcoma (non-Hodgkin's lymphomas). Prolonged Unmaintained Remissions

High doses of methotrexate (HDMTX), given in pulse infusions of 3 to 30 mg/kg body weight, were studied in 22 children with non‐Hodgkins lymphoma. Sixteen complete and five partial remissions were observed in 21 patients evaluable for remission induction. The dose of MTX was increased stepwise on consecutive treatments until objective tumor response occured. Citrovorum factor rescue (CFR) was used “on demand” when toxicity started to develop, and routinely after 30 mg/kg of MTX. Twelve patients who had no previous chemotherapy were entered in a Phase II study consisting of remission induction with HDMTX and remission maintenance with monthly HDMTX supplemented with one monthly injection of vincristine and Cytoxan and five days of oral 6‐mercaptopurine and prednisone. Eleven patients achieved remissions (eight complete and three partial) with HDMTX and one with surgery and radiation followed by HDMTX. The three partial remissions improved to complete remission during remission maintenance. All 12 were given the maintenance cyclic combination chemotherapy. Seven of the 12 patients entered the unmaintained phase of the study. One patient relapsed 6 months after cessation of therapy and died 4 years after diagnosis. Six patients are alive and free of disease 2 1/2 to 5 1/2 years after discontinuing treatment and 4 1/2 to 8 1/3 years after diagnosis. Five of these six patients had advanced (Stage IV) disease.

Immunotherapy for accessible tumors utilizing delayed hypersensitivity reactions and separated components of the immune system.

Courses of repeated delayed hypersensitivity challenge reactions at the sites of tumors have been shown to eradicate malignant and premalignant epidermal neoplasms. Local immunotherapy produces therapeutic responses in malignant and premalignant lesions before they are clinically detectable. This leads to a reduced incidence and prevention of tumors. Immunotherapeutic approaches are effective in controlling the early stages of mycosis fungoides and may aid in the management of the cutaneous manifestations in the late stages of the disease. Immunotherapeutic methods induce regressions of soft tissue lesions of a number of multifocal or metastatic malignant diseases with or without concurrent chemotherapy. Immunotherapeutic effects on tumors are similar with primary and recall antigens. Separated components of the cell-mediated immune system induce regressions of tumors following intralesional or perilesional administration, indicating common factors in host defenses against malignant diseases.

Response of astrocytoma to high‐dose methotrexate with citrovorum factor rescue

Eleven patients with astrocytoma were treated with high‐dose methotrexate (HDMTX) and citrovorum factor rescue (CFR). Clinical response was observed in eight patients, including four of four with grade 3 disease, one of one with grade 2 to 3, two of four with grade four, and one of one with unspecified low‐grade tumor. Two patients with grade 4 and one with grade 1 disease failed to respond. Six of the eight responses were documented radiographically. Three of these patients are alive and well without further treatment for periods of 15+, 4.5+, and 1.5+ years. The fourth living patient is surviving 2+ years and improving under continued treatment. The four surviving patients had recurrent grade 2 to 3 or grade 3 disease. HDMTX‐CFR is effective in astrocytoma. Its greatest value may be in recurring grade 3 disease.

Preliminary observations on tumor regressions induced by local administration of a lymphoid cell culture supernatant fraction in patients with cutaneous metastatic lesions

Abstract A fraction with lymphokine properties was isolated from supernatant medium of the continuous cultured human lymphoblast cell line, 1788. Culture medium containing 2% human serum was used for cell growth in order to minimize antigenicity of isolated supernatant fractions. The culture medium was passed through an Amicon XM-100 membrane, concentrated over a PM-10 membrane, lyophilized and reconstituted to a final concentration of approximately 40:1. Studies in vivo showed that the active fraction contained skin reactive factor which, when injected intradermally into normal skin, produced skin lesions in humans resembling delayed-type hypersensitivity reactions but peaking between 10 and 12 hr. This same preparation, when injected intralesionally into cutaneous tumors, induced an inflammatory reaction followed by tumor regression. Only the fraction confined between membranes of pore size 10,000–100,000 daltons was active in promoting tumor regression. Patients with skin lesions from metastatic carcinoma of the breast and other malignancies were studied, and 16 out of 30 treated lesions were judged to have undergone either complete or greater than 50% regression. Of these, 9 were biopsied before and after lymphokine injection and 6 out of 9 were negative for tumor cells.

Lymphokine properties of a lymphoid cultured cell supernatant fraction active in promoting tumor regression

Abstract In the present study supernatant culture medium from a cultured human lymphoid cell line, which was shown to have inflammation-inducing and tumor-regressing properties in man, was tested for lymphokine properties. Skin reactive factor, lymphotoxin, macrophage activation factor, and chemotactic factor were assayed and found to be present in the crude, active fraction that was confined to a size of 104–105 daltons and was active in producing clinical tumor lesion regression. These results suggest that the in vivo antitumor effect is related to lymphokines present in the active preparation.

The safety of administration of massive doses of methotrexate (50 g) with equimolar citrovorum factor rescue in adult patients

A total of 45 courses of 50 g (24 to 33 g/m2) of high‐dose methotrexate (HDMTX) followed by an improved citrovorum rescue (CFR) were administered to 23 patients according to a recently updated procedure. All patients previously had received HDMTX‐CFR at lower doses. The HDMTX was administered intravenously (IV) over 6 hours with a priming dose of 8 g followed by 42 g given by continuous infusion. Maintenance of adequate urine output and pH level were achieved with IV fluids, sodium bicarbonate, oral acetazolamite, and a low‐acid diet. The CFR was administered by following the equimolar rescue technique and was continued until the serum MTX level was less than 2 × 10−7 mol/l. The MTX was usually rapidly cleared. The median 48‐hour serum MTX level was 7.57 × 10−6 mol/l (range, 6.8 × 10−7 mol/l to 7.9 × 10−5 mol/l). Most patients cleared MTX to below 10−7 mol/l by the eighth day (range, 5 to 13 days) after MTX infusion. The MTX clearance did not always correlate with the pretreatment creatinine clearance. The toxicity observed included the following: leukocyte count less than or equal to 2000/μl in 11% of the courses with less than or equal to 1000/μl in 0%, platelets less than or equal to 105/μl in 9%, creatinine elevation to greater than or equal to 1.5 mg/dl in 7%, mild mucositis without ulcerations in 33%, nausea with occasional vomiting in 66%, mild skin rash in 18%, and temporary elevation of liver enzymes in 81% of the courses. All side effects were tolerable and transient, and the patients recovered fully. Patients who cleared MTX rapidly (MTX ≤ 2 × 10−6 mol/l at 48 hours) rarely sustained leukopenia, creatinine elevation, or skin rash. Toxicity was not increased by third space fluids or by delaying CFR to 24 hours instead of 12 hours after MTX. The procedure described allows the safe administration of HDMTX‐CFR at the 50‐g range to adults with advanced malignant solid tumors.

Breast cancer skin test antigens of increased sensitivity prepared from vesicular stomatitis virus‐infected tumor cells

Crude membrane (CM) extracts were prepared from five cultured breast tumor lines (MDA‐MB‐157, MDA‐MB‐231, ZR75‐1, HS0578T, and MCF‐7) which had been infected with vesicular stomatitis virus (VSV) to augment their antigenicity. In skin test trials, CM extracts of uninfected MCF‐7 cells elicited positive responses in 0 of 13 (0%) tests in breast cancer patients, while VSV‐MCF‐7 elicited positive responses in 11 of the same 13 patients (84.6%). CM extracts of VSV‐ZR75‐1 and VSV‐MCF‐7 elicited greater delayed hypersensitivity responses (mm induration at 48 hr) in breast cancer patients than in patients with lung carcinoma or melanoma. Although the sensitivity of VSV‐ZR75‐1 was too low (ten of 28, or 35.7%, of tests positive) to be useful as a skin test antigen, VSV‐MCF‐7 elicited positive responses in 30 of 38 (78.9%) tests in breast cancer patients, as compared to two of 15 (13.3%) and two of 13 (15.4%) of tests in patients with lung carcinoma and melanoma, respectively. The “virus‐augmented” CM extract of cultured MCF‐7 cells exhibited markedly greater sensitivity as compared to control MCF‐7 extracts (P < .005), with a high degree of specificity for breast cancer patients as compared to patients with the other neoplasms (P < .00001). The results of skin testing with VSV‐MCF‐7 CM extracts demonstrated antigenic cross‐reactivity with a large number of breast cancer patients, a finding of great importance for any potential immunotherapy and/or immunodiagnosis.

Immunotherapy of Skin Cancer and Other Neoplasms Involving the Skin

Various studies have demonstrated that the induction of delayed hypersensitivity reactions at tumor sites results in selective reactions against premalignant and malignant epidermal lesions (Helm and Klein, 1965; Klein, 1967, 1968, 1969; Williams and Klein, 1970). Early lesions which are otherwise clinically undetectable become apparent by their response to immunological challenge, allowing earlier diagnosis and treatment. The more intense delayed hypersensitivity response at the site of premalignant lesions results in their destruction, preventing their subsequent progression to the malignant state.

A Small-Molecular-Weight Growth Inhibitory Factor (SGIF) in Stationary-Phase Supernatants of Tissue Cultures

Tissue cultures stop growing and in time self-destroy when the cell concentration in the medium reaches a certain maximum. This phenomenon has been known for as long as tissue cultures have been used. Periodic addition of medium is a prerequisite for maintaining the cultured cells in adequate growing phase. The common explanation for this phenomenon is that contact inhibition occurs in overgrown cultures. Depletion of nutrients in the culture medium by the growing and metabolizing cells is an alternative explanation. The contact inhibition mechanism was self-suggested by the observation that cells growing in monolayers come in very intimate contact with each other in overgrown cultures, which do not grow further. This explanation of growth arrest was not adequate, in our opinion, to account for the arrest of growth of cells in suspension cultures, such as the human lymphoid cells. In such cultures, the total mass of cells is insignificant compared to the volume of medium in which they float. The depletion of nutrients, on the other hand, is an equally inadequate explanation, for we, and others, have found that cells can grow in media diluted with saline or other solutions for various purposes. We considered, therefore, the hypothesis that the lack of cell growth in stationary-phase cultures is due to the accumulation of materials produced by the cells themselves, which are capable of arresting cell growth when reaching a critical concentration. In fact, the effect of addition of tissue culture medium to keep a cell culture growing may be only the dilution effect on the growth inhibitory factor.