J. Michael Hamilton

Phase I and Pharmacologic Study of 17-(Allylamino)-17-Demethoxygeldanamycin in Adult Patients With Solid Tumors

PURPOSE To determine the clinical toxicities of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) given as a 1-hour infusion daily for 5 days every 3 weeks. PATIENTS AND METHODS Nineteen patients received 17-AAG over six dose levels (10 to 56 mg/m(2)) using an accelerated titration scheme. Drug levels of 17-AAG were determined by high-performance liquid chromatography. Biologic effects of 17-AAG were monitored by changes in the content of target proteins by immunoblot analysis of lysates prepared from peripheral-blood mononuclear cells. RESULTS Toxicity was acceptable at doses up to 28 mg/m(2). The cohort was expanded to three patients at 40 mg/m(2) because a second occurrence of grade 2 hepatic transaminitis occurred. Two of six assessable patients who received 56 mg/m(2) had reversible, grade 3 hepatic transaminitis. Five additional patients were enrolled at 40 mg/m(2); none had dose-limiting toxicity. The maximum plasma concentrations (C(max)) of 17-AAG at 40 and 56 mg/m(2) were 1,724 and 2,046 ng/mL, respectively; the average plasma exposures (AUC) were 2,809 and 6,708 hours.ng/mL, respectively. Less than 3% of the daily dose was excreted into the urine. Clearance did not correlate with body-surface area. Possible biologic activity was suggested by apparent increased protein content of either glucose-related 78 kd protein or heat shock protein 70 with >/= 14 mg/m(2) and decreased protein content of either Lck or Raf1 with >/= 28 mg/m(2) of 17-AAG. CONCLUSION 17-AAG 40 mg/m(2) (median dose, 70 mg) was well tolerated when given daily for 5 days every 3 weeks.

A phase I vaccine trial with peptides reflecting ras oncogene mutations of solid tumors.

Mutations in the ras genes occur in 20% of all human cancers. These genes, in turn, produce mutated proteins that are unique to cancer cells, rendering them distinguishable from normal cells by the immune system. Thus, mutated Ras proteins may form potential targets for immune therapy. We conducted a phase I/pilot clinical trial in patients with advanced cancers to test the toxicity and the ability to induce an immune response by vaccination with 13-mer mutated Ras peptides reflecting codon 12 mutations. These peptides corresponded to each of the patients own tumor Ras mutation. Patients were vaccinated monthly x3 subcutaneously with the specific Ras peptide along with Detox adjuvant (RiBi ImmunoChem Research, Inc., Hamilton, MT, U.S.A.) at one of five different peptide dose levels (100, 500, 1,000, 1,500, and 5,000 micrograms). Three out of 10 evaluable patients generated a mutant Ras specific CD4+ and/or CD8+ T-cell immune response. The CD8+ cytotoxic cells specific for Gly to Val mutation at codon 12 were capable of lysing an HLA-A2-matched tumor cell line carrying the corresponding mutant but not the wild-type ras gene. The treatment has been well tolerated with no evidence of serious acute or delayed systemic side effects on any of the five dose levels. We demonstrated that we can generate in cancer patients specific T-lymphocyte responses that detect single amino acid differences in Ras oncoproteins. Neither the immune responses nor the minor side effects seen were found to be dose dependent. This approach may provide a unique opportunity for generating a tumor-directed therapy. Also, in vitro stimulation of these cells with the corresponding peptide generated specific T-cell lines that could be used for adoptive immune therapy.

The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines.

Abstract An enzyme-linked immunosorbent spot (ELISPOT) assay for interferon γ production has been used to analyze specific T cell responses to a Flu 9-mer peptide, and a 9-mer peptide of carcinoembryonic antigen (CEA). Assays were performed on peripheral blood mononuclear cells (PBMC) of HLA-A2-positive patients with CEA-expressing carcinomas, both before and after vaccination with CEA-based vaccines, and from HLA-A2-positive healthy blood donors. The ELISPOT assay utilized aliquots of frozen PBMC, and assays were performed after 24 h in culture with peptide to rule out any artifacts due to long-term in vitro stimulation cycles. An internal standard was used for each assay to define reproducibility of the assay, and all samples from a given patient (pre- and post-vaccination, with both the Flu and CEA peptides) were analyzed simultaneously. The results indicated a trend towards healthy blood donors having higher levels of Flu-specific T cell precursors than do colon carcinoma patients, but these results were not statistically significant (P = 0.06). On the other hand, slightly higher CEA-specific T cell responses were observed in cancer patients with CEA-expressing carcinomas than in healthy blood donors. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to the Flu control peptide. The first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in the PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific IgG responses were also demonstrated in patients following recombinant CEA poxvirus vaccinations. Statistical analyses of the T cell responses to the same CEA peptide demonstrated a P value of 0.028 for the recombinant poxvirus vaccines, as compared with the peptide vaccine. There were no differences seen (P = 0.37) in Flu-specific responses after these two types of CEA vaccination. These results thus provide the first evidence that poxvirus recombinant-based vaccines are more potent in the initiation of tumor-antigen-specific T cell responses than vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in defining responses to the same peptide. These results also demonstrate for the first time that an ELISPOT assay, performed over a 24-h period and without in vitro sensitization, can be successfully used to monitor immune responses to a tumor-associated antigen in cancer patients.

Strategies for the development of recombinant vaccines for the immunotherapy of breast cancer

SummaryThe development of recombinant vaccines for specific immunotherapy of carcinoma represents a novel approach for the treatment of breast cancer and other tumor types. This article reviews the various parameters that should be considered in the development of recombinant vaccines. Several breast cancer associated antigens are also discussed which may provide potential target molecules. The human carcinoembryonic antigen (CEA), which is expressed on approximately 50% of breast cancers, represents one such target for immunotherapy. To enhance the immunogenicity of this antigen, a recombinant CEA-vaccinia vaccine, designated rV-CEA, was produced. To study the effects of this vaccine in an animal model, a murine colon carcinoma cell line was transduced with CEA and transplanted into immunocompetent mice for protection and therapy studies. Pre-clinical toxicity studies were also conducted in non-human primates. The results of these studies showed the rV-CEA vaccine to be immunogenic and safe in both rodents and primates, and to elicit good anti-tumor responses in the rodent model. In a Phase I clinical trial in metastatic breast, lung, and colorectal cancer patients involving three immunizations of rV-CEA, at three dose levels, enhancement of T-cell and antibody responses to vaccinia virus proteins were observed with no toxicity. Specific T-cell responses were studied via stimulation of peripheral blood lymphocytes with specific peptide epitopes from the CEA molecule. These studies demonstrated clear cut differences in establishment of T-cell lines pre- versus post-immunization. The T-cell lines were shown to be CD8+ and/or CD4+/CD8+, to lyse EBV transformed B-cells transduced with the CEA gene, and to lyse CEA positive carcinoma cells in a HLA restricted manner. Thus, in a Phase I clinical trial the rV-CEA vaccine has been shown to stimulate a CTL response specific for CEA defined epitopes in cancer patients.

Phase I clinical trial of continuous infusion cyclopentenyl cytosine.

Cyclopentenyl cytosine (CPE-C) is an investigational drug that is active against human solid tumor xenografts. The 5′-triphosphate of CPE-C inhibits CTP synthase, and depletes CTP and dCTP pools. We conducted a phase I clinical trial of CPE-C given as a 24-h continuous i. v. infusion every 3 weeks in 26 adults with solid tumors. The starting dose rate, 1 mg/m2 per h, was selected on the basis of both preclinical studies and pharmacokinetic data from two patients obtained after a test dose of 24 mg/m2 CPE-C as an i. v. bolus. Dose escalation was guided by clinical toxicity. A total of 87 cycles were given, and ten patients received four or more cycles. The mean CPE-C steady-state plasma levels (Cpss) increased linearly from 0.4 μM to 3.1 μM at dose levels ranging from 1 to 5.9 mg/m2 per h (actual body weight); the mean total body clearance was 146±38 ml/min per m2. CPE-C was eliminated by both renal excretion of intact drug and deamination to cyclopentenyl uracil in an apparent 2∶1 ratio. CTP synthase activity in intact bone marrow mononuclear cells was inhibited by 58% to 100% at 22 h compared to matched pretreatment samples at all CPE-C dose levels. When all data were combined, flux through CTP synthase was decreased by 89.6%±3.1% at 22h (mean ± SE,n=16), and remained inhibited by 67.6%±7.7% (n=10) for at least 24 h post-CPE-C infusion. Granulocyte and platelet toxicities were dose-dependent, and dose-limiting myelosuppression occurred during the initial cycle in two of three patients treated with 5.9 mg/m2 per h. Four of 11 patients (4 of 20 cycles) who received 4.7 mg/m2 per h CPE-C experienced hypotension 24–48 h after completion of the CPE-C infusion during their first (n=2), third (n=1) and sixth cycles (n=1), respectively. Two of these patients died with refractory hypotension despite aggressive hydration and cardiopulmonary resuscitation. One of 12 patients (28 total cycles) treated with 3.5 mg/m2 per h CPE-C experienced orthostatic hypotension during cycle 1, and this patient had a second episode of orthostatic hypotension at a lower dose (3.0 mg/m2per h). Hypotension was not seen in patients receiving ≤2.5 mg/m2 per h CPE-C. The occurrence of hypotension did not directly correlate with either CPE-C Cpss, CPE-U plasma levels, pretreatment cytidine plasma levels, baseline CTP synthase activity, or with the degree of enzyme inhibition during treatment. While the hypotension appeared to be dose-related, its unpredictable occurrence and the uncertainty concerning the mechanism preclude a recommendation of a tolerable dose for future studies.

Phase I and Pharmacokinetic Trial of Weekly Oral Fluorouracil Given With Eniluracil and Low-Dose Leucovorin to Patients With Solid Tumors

PURPOSE Fluorouracil (5-FU) given as a weekly, high-dose 24-hour infusion is active and tolerable. We evaluated an oral regimen of eniluracil (which inactivates dihydropyrimidine dehydrogenase [DPD]), 5-FU, and leucovorin to simulate this schedule. PATIENTS AND METHODS Patients received a single 24-hour infusion of 5-FU (2,300 mg/m(2) on day 2) with leucovorin (15 mg orally [PO] bid on days 1 through 3) to provide reference pharmacokinetic data. Two weeks later, patients began treatment with eniluracil (20 mg) and leucovorin (15 mg) (PO bid on days 1 through 3) and 5-FU (10 to 15 mg/m(2) PO bid on day 2). RESULTS Dose-limiting toxicity (diarrhea, neutropenia, and fatigue) was seen with 5-FU 15 mg/m(2) PO bid on day 2 given weekly for either 6 of 8 weeks or 3 of 4 weeks, whereas five of seven patients tolerated 5-FU 10 mg/m(2) PO bid given weekly for 3 of 4 weeks. Eniluracil led to a 35-fold reduction in 5-FU clearance. Fluoro-beta-alanine, a 5-FU catabolite, was not detected in plasma during oral 5-FU-eniluracil therapy. DPD activity was markedly suppressed in all patients during eniluracil therapy; the inactivation persisted after the last eniluracil dose; percentages of baseline values were 1.8% on day 5, 4.5% on day 12, and 23.6% on day 19. CONCLUSION The recommended oral dosage of 5-FU (10 mg/m(2) PO bid) given with eniluracil and leucovorin is approximately 115-fold lower than the reference dosage for 24-hour infusional 5-FU. This difference is greater than expected given the reduction in 5-FU clearance. DPD inactivation persisted for several weeks after completion of eniluracil therapy.

A Phase I Dose‐Ranging Study of the Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of AVN944, an IMPDH Inhibitor, in Healthy Male Volunteers

A phase I study of AVN944, an inosine monophosphate dehydrogenase (IMPDH) inhibitor, was carried out to assess its safety, tolerability, pharmacokinetics, and pharmacodynamics. Healthy male volunteers (N = 25) participated in this double‐blind, randomized, placebo‐controlled trial. Sixteen received oral doses ranging from 25 to 250 mg on 3 separate occasions at intervals of 3 or 6 days after overnight fasting. Six participants received two 100‐mg doses, and 2 participants received 2 placebo doses, one with food and the other after fasting overnight. Clinical and laboratory parameters, including excretion of AVN944 and thiocyanate and IMPDH inhibition, were made at intervals for 48 hours postdosing. There were 13 mild and 2 moderate but no serious adverse events. One mild and 1 moderate event could be treatment related. AVN944 disappeared rapidly from plasma, but clearance decreased at doses >50 mg. Food reduced absorption, with a geometric mean Cmax ratio of 33% and a geometric mean AUC0‐∞ ratio of 44%. Urinary excretion was negligible. AVN944 doses >100 mg showed definite IMPDH inhibition lasting at least 4 to 6 hours. AVN944, when administered orally to healthy volunteers, is well tolerated, absorbs better with fasting, and exhibits a pharmacodynamic profile that suggests potential for significant anticancer activity.

Preliminary Pharmacokinetics (PK) of COL‐3, A Matrix Metalloproteinase (MMP) Inhibitor

Clinical Pharmacology & Therapeutics (1999) 65, 195–195; doi:

Varicella zoster meningitis preceeded by thrombophlebitis in a patient with Hodgkin's disease.

Varicella zoster (V-Z) infections are common among patients with hematological malignancies, particularly Hodgkins diease (HD). The common denominator in both HD and V-Z infections is immunosuppression. Most of V-Z infections occur in patients with HD during the remission period, who have mixed cellularity sub-type. with stage III disease and who have received combined chemo-radiation therapy. Involvement of the control nervous system usually manifests as post-herpetic neuralgia or encephalitis. Angiitis has also been found in association with V-Z infections. The authors describe a case of HD who developed V-Z meningitis preceeded by superficial thrombophlebitis of upper extremities during the period of active chemotherapy.