Goralatide (AcSDKP), a negative growth regulator, protects the stem cell compartment during chemotherapy, enhancing the myelopoietic response to GM-CSF.

The aim of our study was to investigate the protection afforded to the bone marrow by Goralatide (AcSDKP), an inhibitor of hemopoietic stem cell proliferation, when administered alone or in combination with a growth factor (granulocyte/macrophage colony-stimulating factor [GM-CSF]) during iterative cycles of Ara-C (cytarabine) treatment. In control mice receiving the inhibitor alone without Ara-C, the number of granulocytes was reduced during treatment, and a surge in number of peripheral blood cells was observed after its completion. Peripheral hematological responses were monitored during 3 consecutive cycles of Ara-C chemotherapy and the resultant nadir and recoveries. Analysis of variance of the treatment effects pooled over the 3 cycles showed that a treatment regimen in which the inhibitor was administered during the myelotoxic periods of chemotherapy confirmed the existence of a surge after completion of administration of the inhibitor and showed a significant protective effect. When the cycles of chemotherapy plus Goralatide were followed by GM-CSF, the recovery from leukopenic nadirs was accelerated and the white blood cells and granulocyte levels were markedly increased over those observed in control mice and in mice treated either with Goralatide alone or with GM-CSF alone. The differences were highly significant. A consistent and significant increase (p < 0.001) in platelet count was also noted in animals given Goralatide in conjunction with Ara-C or Ara-C + GM-CSF. After three treatment cycles, this response to the CSF was far better in mice treated by the inhibitor than when CSF was given alone, suggesting a protection of the stem cell pool.

Proliferative response of human and animal tumours to surgical wounding of normal tissues: onset, duration and inhibition.

Acceleration of secondary tumour growth and metastases following excision of a primary tumour has been attributed to the consequent removal of primary tumour-generated inhibitory factors. However, our studies have shown that surgical wounding of normal tissues significantly stimulated the growth of malignant tissues without the concomitant presence or excision of a tumour mass. A humoral stimulating component was indicated by the proliferative response of tumours and metastases distant from the surgical wound. All 16 human and murine tumours, of nine different histologies, showed a measurable acceleration of growth when implanted in surgically treated animals, suggesting that the ability of malignant tissue to respond to surgical wounding of normal tissue was not histologically or species specific. The proliferative surge of malignant tissues was detectable soon after wounding and had a duration of 2-3 days. The surgical wound as the source of the tumour-stimulating factor(s) was affirmed by the significant inhibition of tumour proliferative responses when a somatostatin analogue was applied topically to the surgical wound within 1 h of wounding, and/or during the critical tumour-stimulatory period of 1-2 days after wounding. A potential therapeutic window for reducing a risk factor that may be inadvertently imposed upon every surgical/oncology patient is indicated.

Proliferative response of human prostate tumour xenografts to surgical trauma and the transurethral resection of the prostate controversy.

Transurethral resection of the prostate (TURP) as an excisional procedure involving multiple incisions into the prostate does not differentiate between palpably benign prostate tissue and microscopic foci of well-differentiated adenocarcinoma. The impact of TURP on the progression of such 'latent' or 'incidental' tumours unique to the prostate gland has been a focal point of a continuing controversy. In studies designed to develop preclinical evidence that would lend support to, or detract from, either side of the TURP controversy, surgical trauma-induced stimulation of in situ tumour growth was extended to include human prostate tumour tissue PC-3, DU-145 and H-1579, albeit as xenografts in athymic nude males. A significant proliferative response of prostate tumours implanted directly in, adjacent to, or distant from, a freshly induced surgical wound, could be inhibited by a somatostatin analogue (Lanreotide) applied topically to the surgical site. This preclinical model supports TURP as a risk factor for biopsy or therapeutic surgical intervention procedures in benign prostatic hypertrophy (BPH), a risk factor that increases with the stage of disease in undetected cancers. It also suggests a potential clinical benefit that might be derived by applying Lanreotide directly to the surgically traumatised genitourinary area by simple irrigation of the urethra and bladder during or shortly post TURP.

Systematic development of bombesin/gastrin-releasing peptide antagonists.

Several families of very potent bombesin (Bn) receptor antagonist analogues have recently been developed and their biological potencies evaluated in a number of in vitro systems including guinea pig and rat pancreatic acini and Swiss 3T3 cells. These studies showed that analogues can exhibit diverse properties ranging from full antagonists, partial agonists, or full agonists depending on the assay system and animal species employed. We have developed two classes of more potent, shorter chain antagonists based on [psi CH2NH(13-14)]Bn(6-14) and desMet14Bn(6-13)NH2 structures. [D-Phe6 psi Leu13-Leu14] Bn(6-14)NH2 was a potent antagonist (Ki 6nM) in Swiss 3T3 cells and guinea pig acini but exhibited 10% partial agonist activity and lower binding affinity (Ki 60 nM) in rat acini. The partial agonism could be eliminated by using p-Cl-Phe or D-Phe at the C-terminus and partially eliminated using D-4-Cl-Phe in position 6. With the antagonist [D-Phe6]Bn(6-13)NH2 (Ki 96 nM), alkyl substituents on the amide group increased affinity 25-fold with the propylamide being the most potent peptide (Ki 4 nM) in 3T3 cells or guinea pig acini. It did, however, have high 40% partial agonist activity in rat acini. Alkyl esters or hydrazide derivatives were, in contrast, pure antagonists in all systems tested with [D-Phe6]Bn(6-13)OMe having the highest affinity in all systems and also excellent in vivo properties. All of the potent antagonists examined had little affinity for neuromedin B--preferring bombesin receptors, which had entirely new ligand structure-activity relationships.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of human lung tumor xenografts to treatment with a somatostatin analogue (Somatuline).

Four human small cell lung carcinomas, NCI-H69, NCI-N417, NCI-H345, LX-1, and a non-small cell lung carcinoma, H-165, implanted s.c. as tumor xenografts in athymic nude mice, were treated with Somatuline (BIM-23014C), an endocrinologically potent octapeptide analogue of somatostatin. All tumors responded, although in varying degrees, with percentage of test/control values ranging from 3 to 88. Somatuline administered as a perilesional infusion effectively inhibited xenograft growth inducing prolonged remissions. When treatment was terminated, some tumors regrew, suggesting antimitogenic activity rather than cytocidal. Absence of observable systemic or local toxicity during prolonged treatment would support this conclusion and suggest the feasibility of long term maintenance therapy with a resultant extended survival.

Treatment of R-3327 prostate tumors with a somatostatin analogue (somatuline) as adjuvant therapy following surgical castration.

This study addresses, in an animal tumor model, the clinical problem of "escape from castration inhibition." Somatuline (BIM-23014C), an octapeptide analogue of somatostatin with enhanced potency and longer duration of biological activity was administered as a therapeutic agent, over a period of 90 and 197 days, to male Copenhagen rats bearing syngeneic Dunning R-3327-H prostate tumors. Androgen sensitivity was confirmed by the response of tumors to castration and by the significant inhibition of tumor growth in intact animals by treatment with a luteinizing hormone-releasing hormone antagonist (BIM-21009). Inhibition of tumor growth resulting from castration persisted for 102 days, after which progressive regrowth occurred, indicating an escape from castration inhibition. When Somatuline treatment was initiated as an adjuvant therapy 5 days after castration, the rate of tumor regrowth during escape was significantly retarded. During the period of 197 days postcastration, tumors in the vehicle-treated, intact controls grew to an average diameter of 38.6 +/- 7.6 mm and tumors in vehicle-treated castrate controls grew to an average diameter of 23.3 +/- 4.1 mm (60% test/control). Treatment with the luteinizing hormone-releasing hormone antagonist induced no significant additional tumor inhibitory effects in castrated animals which developed tumors having an average diameter of 30.2 +/- 8.2 mm (78% test/control). Treatment of tumors in castrate animals with Somatuline, on the other hand, induced a significant (P less than 0.01) tumor-inhibitory effect that was greater than that produced by castration alone, developing an average tumor diameter of only 14.3 +/- 2.6 mm, (37% test/control). A growth inhibitory effect was also inducible in animals having tumors that had already escaped castration inhibition. The relative nontoxicity of a somatostatin analogue such as Somatuline suggests that chronic or maintenance therapy of slow-growing prostate cancers may be both feasible and acceptable in a clinical setting.

In vitro and in vivo inhibition of human small cell lung carcinoma (NCI-H69) growth by a somatostatin analogue.

An endocrinologically-potent octapeptide analogue of somatostatin (SRIF), 3-(2-naphthyl)-D-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (BIM-23014 C), was examined for its ability to inhibit the in vitro and in vivo growth of the human small cell lung carcinoma (SCLC) line, NCI-H69. When cultured cells were implanted into athymic nude mice, treatment (500 micrograms/injection, twice daily) resulted in a prolongation of lag time for the appearance of measurable tumors, and there was a marked inhibition of the growth rate. Indeed, peptide injection in the region of the tumor resulted in a complete regression of the NCI-H69 tumors. Withdrawal of BIM-23014 C treatment resulted in an acceleration of tumor growth indicating an antiproliferative rather the oncolytic action. A similar inhibition of tumor growth was also observed when solid tumors obtained from the first implantation were used as the donor tissues. In cell culture, the proliferation in the presence of a low concentration (10nM) of BIM-23104 C was also significantly retarded suggesting a direct mechanism of action.

The subrenal capsule assay (SRCA) and its predictive value in oncology.

This report has been prepared as a mini-review of the studies and data, accumulated by a number of investigators since 1978, that are relevant to the 6-day subrenal capsule assay (SRCA) as a predictive in vivo test system. Stressing the need to maintain simplicity and economy, the data reviewed are based on the use of normal, immunocompetent mice and a simple tumour size parameter for evaluating drug activity within a 6-day time frame. Both the laboratory and clinical data that are presented and evaluated support the 6-day SRCA as a predictive test system at the preclinical and clinical levels of drug development and treatment. The question, do host responses create an artifact in the tumour size parameter, is addressed with experimental data illustrating the sensitivity of tumour xenografts, prepared from endocrine target tissues, to respond to biological effects in the 6-day SRCA. SRCA data are also presented suggesting that tumour heterogeneity does not automatically preclude the usefulness of predictive assays based upon a tumour sample.

Subrenal capsule assay to test the viability of parenterally delivered müllerian inhibiting substance.

Production of bovine müllerian inhibiting substance (MIS) has been increased to allow generation of large quantities of biologically active purified material. The limited MIS previously available allowed only pretreatment of tumors prior to colony inhibition or implanting in nude mice. In preparation for posttransplantation tumor treatment, a subrenal capsule assay, which was first used against human tumors heterotransplanted into nude mice and subsequently against those heterotransplanted into immunocompetent mice, was adapted to determine (1) if MIS preparations could traverse the bloodstream without degradation and (2) the optimal dose required to produce a biologic effect. Urogenital ridges from female 14-day-old rat embryos were transferred atraumatically to small pouches beneath the renal capsule of the immunocompetent male CDF1 mice. The cranial-caudal orientation of the ridge with its müllerian duct was maintained. Over the next 72 hours, the mice were injected via the tail vein with 0.1 mL of an MIS-containing solution over a 100-fold concentration range. After three days, the kidneys were removed and shaved just below the ridge, which was then placed in soft agar for orientation and subsequent serial sectioning. After fixation, dehydration, and paraffin embedding, sections were stained and regression of the müllerian duct was graded and compared according to concentration and number of MIS doses administered. Regression diminished from almost complete (4+) at the highest dose, to minimal (1 to 2+) at 1/100 of that dose. Heat-inactivated and vehicle controls caused no regression of the müllerian ducts.(ABSTRACT TRUNCATED AT 250 WORDS)

9-Deazaadenosine--a new potent antitumor agent.

-Deazaadenosine (9-DAA), a novel purine analog, was found to be a potent inhibitor of the growth of nine different human solid tumor cell lines in vitro and of pancreatic carcinoma (DAN) in antithymocyte serum (ATS)-immunosuppressed mice. In culture, IC50 values ranged from 1.1 to 8.5 X 10(-8)M. Ovarian carcinoma (MR) was the only cell line in which the activity of 9-DAA was potentiated (about 10-fold) by pretreatment with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF). After incubation of cultured pancreatic DAN cells with 9-DAA (10(-5)M) for 2 hr, a peak appeared in the triphosphate region of HPLC nucleotide profiles that was identified tentatively as 9-deazaATP. Under the same incubation conditions, the incorporation of [3H]uridine into RNA and of [3H]thymidine into DNA was inhibited by 34 and 80% respectively. In vivo studies using ATS-immunosuppressed mice showed that 9-DAA at 0.4 mg/kg/day for 3 consecutive days reduced pancreatic carcinoma (DAN) tumor weights to approximately 50% of untreated controls. The nucleoside transport inhibitor p-nitrobenzyl-6-thioinosine (NBMPR) was shown to selectively protect host tissues from 9-DAA toxicity and, thereby, potentiated the antitumor activity of 9-DAA in vivo at optimal dosages.

Comparison of the human tumor cloning and subrenal capsule assays.

Fresh surgical explants of solid tumors obtained from 84 patients were tested against the same chemotherapeutic agents in both the in vitro human tumor cloning (HTC) assay and the in vivo subrenal capsule (SRC) assay. Control growth adequate to meet evaluable assay criteria was obtained in 75 of 84 tumors tested in the SRC assay (89%) and in 33 of 79 tumors tested in the HTC assay (42%). Correlations between the two test systems were dependent upon the activity criteria established for each system. With activity criteria set at current drug screening levels as a decrease of greater than or equal to 50% in tumor colony-forming units for the HTC assay and a change in tumor size less than -1.0 ocular micrometer unit for the SRC assay, 16% of the drugs tested were active in the SRC assay, and 7% were active in the HTC assay. Correlations of tumor response between the two assays were 29% for sensitive (2 of 7) and 83% for resistant (63 of 76). Of the 84 patients providing tumor tissue for assay, 17 had clinically evaluable disease and received chemotherapy providing information for retrospective analysis. A total of 23 SRC assay-clinical correlations and 10 HTC assay-clinical correlations were possible. The SRC assay was predictive of clinical sensitivity in three of three drug tests (100%) and of clinical resistance in 16 of 20 drug tests (80%). No HTC assay-clinical correlations were possible for sensitivity, but the HTC assay was predictive of clinical resistance in 10 of 10 drug tests (100%).

Predictive testing with the subrenal capsule assay.

Measurement of drug activity as an oncolytic effect, use of the control only to monitor the quality of tissue implanted, and the rapid clearance of necrotic tissue from the subcapsular site, as significant factors incorporated into the design of the assay, have permitted use of a simple tumor size parameter for evaluating drug activity. The simplicity and economy of such a parameter, the predictability and reproducibility of the 6-day assay observed thus far, and evidence that the assay does measure a biological property of the tumor apart from host response, have warranted the continued use of the 6-day time frame and the normal immunocompetent CDF1 mouse as xenograft host. These studies have demonstrated the feasibility of using human tumor explants obtained from a variety of solid human malignancies in a straightforward, short term, in vivo predictive assay system. Preliminary correlations between in vivo (assay) tumor sensitivity and clinical response have given reasonable concurrence. This crucial point will require further study, with larger numbers of patients, under more rigid conditions. Final validation of this, and other, predictive assays will require a prospective, randomized study in large numbers of patients. Our present prospective study is being continued, therefore, with expansion to a multi-institutional design over a broader geographic area.

Initial clinical trials of the subrenal capsule assay as a predictor of tumor response to chemotherapy.

Retrospective and prospective clinical trials were performed to determine the usefulness of the 6-day subrenal capsule (SRC) assay for the prediction of response to chemotherapy. Evaluable assays were obtained in 86% of 1000 consecutive specimens obtained from a variety of solid malignancies. Analysis of chemotherapeutic sensitivity in this assay gave reproducible and consistent results. The overall predictive accuracy of the assay in 62 retrospective clinical trials in 55 patients was 85%. Of 37 evaluable patients with chemotherapy refractory cancers treated in a prospective trial with single agent chemotherapy as determined by the assay, 14 (38%) responded. Greater degrees of tumor regression in the assay were associated with a higher probability of clinical response. The SRC assay shows potential value as a rapid predictive test for chemotherapeutic selection on an individual patient basis. However, additional prospective clinical trials are necessary to document its ultimate utility.

Chemotherapy responsiveness of human breast tumors in the 6-day subrenal capsule assay: an update.

Feasibility of utilizing the 6-day subrenal capsule (SRC) assay to screen drugs against fresh surgical explants of human tumors was confirmed by testing six clinically active chemotherapeutic agents against 141 human breast cancers. Response rates of the six drugs obtained in the assay compared favorably with clinical response rates for the same drugs as reported by Carter (5). The evaluable assay rate for breast tumors was 92% as compared to 89% for gynecologic tumors. Innate drug resistance was indicated with 16 of 57 tumors (28%) which did not respond to any of the six agents tested. Differences in responsiveness of tumors to each agent in a potential three-drug combination of either CMF or CAF suggest that the effectiveness of multiagent therapy might be enhanced if the individual agents of a potential drug combination were selected on the basis of tumor sensitivity to each of the agents in a predictive assay. Although cross-resistance between L-PAM and cytoxan was demonstrated and was statistically significant, 31% of these tumors responded individually to either one or the other agent, suggesting caution in extrapolating concomitance in activity between these two alkylators.

Activity of two phase I drugs N-methylformamide (NSC-3051) and Echinomycin (NSC-526417) against fresh surgical explants of human tumors in the 6-day subrenal capsule (SRC) assay.

The potential clinical activity of the new phase I drugs N-methylformamide (N-MF) and Echinomycin (ECH) was examined while still undergoing clinical toxicology trials by testing against fresh surgical explants of human tumors in the 6-day in vivo SRC Assay. Sixty-nine tumors representing different histologic types including breast, lung, colon, ovarian, and cervical, as well as neoplasms of undiagnosed origin, were screened against N-MF (NSC-3051) and ECH (NSC-526417) simultaneously with five standard chemotherapeutic agents used clinically for treatment of the specific type of cancer. Thus, activity of N-MF and ECH could be compared directly with that of standard agents tested in the same assay. Treatment schedule was QD1-5, and the criterion for drug activity was tumor graft regression greater than 20%. N-MF was active against 15/69 tumors with a response rate of 22%. ECH was also active against 15/69 tumors, yielding the same response rate. Although the response rates for N-MF and ECH were the same, indicating a similar degree of general anti-tumor activity as evaluated by the assay, N-MF showed greatest activity against lung tumors whereas ECH was more active against ovarian tumors. Twenty-six of 69 tumors (38%) were unresponsive to all drugs tested, only one tumor was responsive to both N-MF and ECH and no tumors were responsive to either N-MF or ECH alone. Cytoxan, one of the standard agents tested concurrently with both phase I drugs yielded a response rate of 35%, one and one-half times greater. Cervical and renal cancers and lymphomas were relatively unresponsive to both drugs.