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Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple "smash" technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.

Advanced controlled release (CR) dosage forms are relative newcomers to pharmaceutical markets, and few studies relate their efficacy, safety or compliance benefits to economic value. This literature review was undertaken to assess the cost effectiveness of CR dosage forms using such measures as purchase costs, total treatment costs, and economic value of improved therapeutic outcomes compared with those with non-CR dosage forms. Three therapeutic areas were examined: cardiovascular therapy, pain management and estrogen replacement therapy. In cardiovascular therapy, prescription costs of sustained release (SR) verapamil were significantly higher than for conventional release verapamil. However, these were more than offset by lower physician, hospital and laboratory expenditures for the SR group, in whom compliance was superior. Similarly, patients receiving SR diltiazem had better prescription refill compliance than those using a conventional formulation of the drug, as well as significantly lower aggregate healthcare costs during a 1-year study period. These lower costs with both SR verapamil and diltiazem may relate to better compliance. CR nifedipine has lower daily acquisition costs than the conventional form, simplifies the dosage regimen to once daily, extends the indications of the drug to hypertension as well as angina, and reduces vasodilatory adverse effects by reducing peak plasma drug concentrations and the postdose rate of increase in concentration. Compared with oral clonidine given twice daily, transdermal clonidine, given once weekly, had higher purchase costs, but was associated with improved compliance, reduced adverse effects (due to control of plasma concentrations), and lower nondrug health costs, such as physician, hospital and laboratory costs. Lower costs were also found for once daily oral formulations of various antihypertensives, suggesting that the economics of both types of CR dosage forms related to compliance. CR metoprolol 50 or 100mg and conventional release atenolol 50mg, each given once daily, provided effective beta1-adrenoceptor blockade. The conventional formulation caused deterioration in the sense of well-being that was temporally associated with sharp peaking of its plasma concentrations. Such peaking did not occur with either dose of CR metoprolol, nor did any deterioration in the sense of well-being. Transdermal nitroglycerin (glyceryl trinitrate), compared with long-acting oral nitrates, improved quality of life (QOL) {despite a higher incidence of some adverse effects, such as headache, dizziness and skin irritation}. Furthermore, in some studies, this formulation reduced angina attacks, sublingual nitroglycerin use, and hospitalisation or emergency room use. Cost comparisons between transdermal products favoured those that have superior adhesion.(ABSTRACT TRUNCATED AT 400 WORDS)

The application of recombinant DNA technology to the production of tumor necrosis factor has resulted in the availability of large quantities of a highly purified protein product. This product has been evaluated extensively in preclinical studies, which have documented a direct cytostatic and cytotoxic effect on human tumor cells, as well as a variety of immunomodulatory effects on various immune effector cells, including neutrophils, macrophages, and T cells. In addition, a number of anti-infective and metabolic effects have been documented. In addition to its in vitro effects, rTNF has been shown to have antitumor activity in vivo in preclinical studies involving both transplantable murine tumors and human tumor xenografts. Such observations have led to the evaluation of rTNF as a potential antineoplastic agent in humans. Both single- and multiple-dose phase I studies have confirmed that rTNF can be safely administered to patients with advanced malignancies in a dose range associated with anticancer effect without concomitant serious toxicities such as shock and cachexia. The most commonly observed clinical toxicities include constitutional symptoms, such as fever, chills, headache, and fatigue, and toxicities, which can be at least partially controlled with concomitant administration of nonsteroidal anti-inflammatory drugs, such as acetaminophen and meperidine. Hypotension, which occurs at high doses administered by short intravenous infusion, can usually be prevented by prehydration with intravenous fluids or otherwise controlled by the administration. An intense local inflammatory reaction at the injection site as well as thrombocytopenia appear to be the dose-limiting toxicities after subcutaneous and intramuscular administration. Neurologic toxicity is infrequent, except following continuous intravenous infusion, where it may manifest as transient focal neurologic deficits or seizure. Prolonged administration of rTNF at higher doses may be associated with transient, subclinical decreases in diffusing capacity. Patients with underlying cardiopulmonary disease should be excluded from rTNF therapy in future clinical studies until the end-organ toxicities of this agent are better defined. For at least one preparation of rTNF there appears to be no evidence for the formation of antibodies to rTNF in patients who receive multiple administrations of the agent. Pharmacokinetic studies have shown a relatively rapid clearance following intravenous infusion with a half-life of 15 to 30 min and dose-dependent pharmacokinetics. rTNF can be detected in the serum following intramuscular or subcutaneous injection at only relatively high doses, suggesting a decreased bioavailability with the routes of administration. Early phase I studies defined tolerable dose ranges for each route of administration and began to explore immunomodulatory and metabolic effects of rTNF.(ABSTRACT TRUNCATED AT 400 WORDS)

Recombinant human tumor necrosis factor and recombinant human interferon-gamma are two representatives of a novel class of antineoplastic agents. Evaluation of these agents in vitro has suggested that the combination would be more effective than either agent alone. A prior phase I study demonstrated that the maximum tolerated dose for each agent was 150 micrograms/m2/day for 5 days when administered concomitantly. Based on this experience, a phase II trial of patients with biliary tract, pancreatic, and colorectal cancer was planned. Our goal was to treat a minimum of 14 patients with each tumor type. However, in the first 13 patients entered into this trial the toxic effects at the starting doses of 125 micrograms/m2/day for 5 days for each agent were intolerable, with four patients unable to complete planned therapy. In this cohort of patients, no objective responses were observed. Further clinical investigation of this combination should consider alternative treatment schedules to reproduce the in vitro synergistic cytotoxicity of this combination while minimizing host toxicity.

The proto-oncogene HER2/neu encodes a protein tyrosine kinase (p185HER2) that is homologous to the human epidermal growth factor receptor. Amplification and/or overexpression of HER2/neu occurs in multiple human malignancies and appears to be integrally involved in progression of some breast and ovarian cancers. Because of this fact, HER2/neu is an intriguing target for specific cancer therapeutic strategies. One such strategy is active specific immunotherapy, in which the immune system is targeted at specific antigens expressed by tumor cells. We have employed a transfected cell line that secretes the extracellular domain of p185HER2 as a source of HER2-derived immunogen in a guinea pig model. The immunized animals developed a cellular immune response, as monitored by delayed-type hypersensitivity, and antisera derived from immunized animals specifically inhibited the in vitro growth of human breast tumor cells overexpressing p185HER2. These data provide support for an immunotherapeutic approach to cancers characterized by overexpression of the HER2/neu proto-oncogene.

Recombinant human interferon gamma (rIFN-gamma) was used for the treatment of 16 patients with various stages of cutaneous T-cell lymphoma (CTCL). All patients had been previously treated with standard topical and/or systemic therapies, and some had received experimental treatment with retinoids, recombinant human interferon alfa-2a (rIFN-alpha 2a), or radiolabeled monoclonal antibodies; most patients had an advanced stage of disease. Objective partial responses (PRs) were noted in five patients (31%) and lasted 3 months to greater than 32 months (median, 10 mo). One of these five patients had previously had disease progression after an initial PR with rIFN-alpha 2a. Six other patients (38%) showed minor or mixed responses. The most common side effects of rIFN-gamma included fever, weight loss, mild neutropenia, elevated lactate dehydrogenase, and elevated hepatic transaminases. Additionally, one episode of nephrotic syndrome and one cutaneous allergic reaction were noted. None of the toxic effects were life threatening, and all were reversible. These results suggest that rIFN-gamma has efficacy in the treatment of CTCL refractory to rIFN-alpha 2a.

Tumor necrosis factor (TNF) has demonstrated antitumor activity against a variety of tumors and is particularly cytotoxic to capillary endothelial cells, which are the presumed cell of origin of Kaposi's sarcoma. We evaluated the toxicity and clinical antitumor and antiretroviral effects of recombinant TNF administered at a once weekly dose of 100 micrograms/m2 intravenously for 8 weeks in five men with AIDS-related Kaposi's sarcoma and without prior opportunistic infection. One patient was removed from study at week 4 due to rapid progression of Kaposi's sarcoma, another patient with stage IV disease and a pretreatment CD4 count of 11 developed fever, hypotension, and pneumonia at week 7 and died 8 days after discontinuing recombinant TNF. No pathogenic organisms were isolated. He had marked eschar formation of his Kaposi's sarcoma lesions, particularly in areas previously exposed to radiation therapy. Uniform toxicities included fevers, rigors, and headaches during drug infusion that were ameliorated by prophylactic meperidine hydrochloride and acetaminophen. All experienced fatigue and three had arthralgias. One patient had transient hypotension which corrected with i.v. fluids. No significant hematologic, hepatic, or renal toxicities were seen. All patients had some progression of their Kaposi's sarcoma on study. There was no change in CD4 or CD8 count or in CD4:CD8 ratios. Serum human immunodeficiency virus (HIV) p24 antigen levels increased greater than 50% in three patients. We conclude that, as a single agent, at a dose of 100 micrograms/m2 recombinant TNF by i.v. infusion has no obvious antitumor or antiretroviral effects in patients with AIDS-related Kaposi's sarcoma.

Preclinical studies have suggested that synergistic antitumor toxicity occurs when methotrexate (MTX) is administered prior to 5-fluorouracil (FUra). A protocol of sequenced, overlapping infusions of MTX and FUra was designed to achieve 5 microM MTX serum levels lasting 36 h and 1 to 5 microM FUra levels lasting 24 h, with leucovorin started at the end of the MTX infusion. Thirty-nine patients with metastatic neoplasms received a total of 127 treatment courses; two-thirds of the patients had received prior treatment with radiation therapy or chemotherapy; most of the latter treatment regimens included MTX or FUra. In three patients, the duration of FUra infusion was prolonged up to 72 h to determine the toxic limits of therapy. Blood samples were collected during treatment courses to estimate the half-lives and total-body clearances of MTX and FUra. The initial serum half-lives and total-body clearances of both MTX and FUra appeared within the range of reported normal values. The terminal half-life of MTX appeared less than previously reported values, and there appeared to be a substantial delay in achieving a FUra steady-state concentration; these two differences may have resulted from either the prolonged intervals of drug infusion or from metabolic interaction between the two drugs. During the 127 courses of treatment, nearly one-half of the patients experienced mild toxicity occurring after at least one treatment, but this toxicity was predominantly Grade I mucositis and/or diarrhea. Of the three patients who received extended intervals of FUra infusion, none was able to tolerate more than 48 h of FUra without developing mucositis. Thirty-four patients were evaluable for response; no one experienced a complete response, but 11 (32%) patients had either a partial or minimal response. Adenocarcinomas as a group, arising from the lung, gut, breast, and unknown site, appeared to respond best. Sequenced MTX-FUra infusion by this schedule is a generally well-tolerated regimen that deserves further clinical assessment.

Combinations of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) demonstrate synergistic antiproliferative activity in vitro. Therefore, we initiated a clinical study of recombinant TNF-alpha (rTNF-alpha) and rIFN-gamma combination therapy in humans. Twenty-five patients with metastatic cancer received both rTNF-alpha and rIFN-gamma by intramuscular injection for 5 consecutive days every 2 weeks for a total of 4 weeks. The dose levels were 5/5, 10/5, 10/10, 25/10, 25/25, 50/25, 50/50, 75/50, and 75/75 micrograms/m2 per day of rTNF-alpha/rIFN-gamma. A minimum of 2 patients were entered sequentially at each dose level. If the first 2-week cycle of therapy was well tolerated, the dose was increased to the next highest dose level during the second 2-week cycle. Fever, chills, and fatigue were observed at all doses. Severity of symptoms corresponded to increasing dose levels. Although TNF is identical to a molecule known as "cachectin," the vast majority of our patients did not lose weight while on study. However, alterations in lipid metabolism occurred and were manifested by a median change in triglyceride values of +40 mg/dl (range, -130 to +318 mg/dl), and in cholesterol values of -30 mg/dl (range, -103 to +2 mg/dl). The maximum tolerated dose was 75 micrograms/m2 of rTNF-alpha combined with 50 micrograms/m2 of rIFN-gamma, with dose-limiting side effects being mainly constitutional symptoms. A dose-related suppression in granulocyte and platelet counts was observed. Hematologic parameters returned to baseline within 72 h after therapy was discontinued, and neither infection nor bleeding occurred. Ten of 22 evaluable patients had stable disease for a median of 8 weeks (range, 4-21 weeks); 12 patients showed progressive disease. This study will form the framework for phase II trials of rTNF-alpha and rIFN-gamma combination therapy.

The effect of recombinant tumor necrosis factor-alpha (rTNF), injected directly into the tumor, was evaluated in a Phase I/II study of 27 patients with AIDS-associated Kaposi's sarcoma (KS). The maximally tolerated intralesional dose was less than 100 micrograms/m2 and the recommended intralesional dose was 25 micrograms/m2. In a double-blind, randomized, placebo-controlled study, rTNF reduced the cross-sectional area of 15 of 16 (94%) of the injected KS lesions and caused complete disappearance of 3 of 16 (19%) lesions. Only injected lesions showed a response. Rigors and fever were common dose-dependent side effects and were attenuated by meperidine. There were no changes in human immunodeficiency virus (HIV) activity as determined by serum p24 antigen levels. While biologically active, the systemic toxicity of rTNF as well as the lack of distant antitumor effects in noninjected lesions limits its clinical usefulness under the conditions employed in this trial.

The etiologic relationship of the Epstein-Barr virus (EBV) to nasopharyngeal carcinoma prompted this study of the efficacy of interferon-gamma (IFN-gamma), which possesses antiviral and antitumor activity, in the treatment of recurrent carcinoma. Fourteen patients received recombinant IFN-gamma in daily intramuscular doses of 5-10 X 10(6) U/m2. Of 13 patients evaluable for response, five had a minor response; disease was stable in two and had progressed in six. EBV serology for immunoglobin (Ig) G, IgA-VCA, and early antigen was measured in six patients. In four of these, titers rose consistently with disease progression and in one patient, titers decreased with minor response.

Fifteen patients with multiple myeloma, five with hairy cell leukemia, and five with Waldenstrom's macroglobulinemia were treated with recombinant interferon gamma (rINF-gamma) to determine the antitumor activity of this agent. The rIFN-gamma was administered by daily intramuscular injection at doses ranging from 0.125 to 0.5 mg/m2. No responses were observed in patients with multiple myeloma, although in one patient the disease has remained stable for over 16 months. Minimal improvement in some hematologic indexes were observed in three of five patients with hairy cell leukemia. One partial remission and one minor response were documented in two of the five patients with Waldenstrom's macroglobulinemia. In five patients, an increase in normal serum immunoglobulins was observed. These results suggest that there is only minimal activity of rIFN-gamma as a single agent in neoplasms of B-cell origin.

A sensitive and precise ELISA has been developed for the quantitation of recombinant Tumor Necrosis Factor-Alpha (rTNF-alpha) in undiluted sera. Affinity purified rabbit antibody was used as capture antibody and mouse monoclonal antibody labelled with horseradish peroxidase was used as the second antibody in a sandwich ELISA. The assay range was from 50 to 2000 pg/ml and the relative standard deviation was 8% or less for both interassay and intra-assay precision studies. Recovery of rTNF-alpha added to 10 different human and 10 different monkey sera ranged from 81 to 102% and 100 to 120% of the expected value, respectively. This ELISA has been used to measure serum rTNF-alpha levels in over 60 patients in Phase I Clinical Trials treated with rTNF-alpha. The levels in a representative, pharmacokinetic study showed low variability between 8 patients receiving intravenous bolus administration of 100 mu rTNF-alpha/m(2). The ELISA results correlated well with TNF bioassay data with a mean specific activity of 2.5 x 10(7) U/mg.

Fifty-three patients with metastatic-renal cell carcinoma received treatment with combinations of recombinant alpha (rIFN-alpha-2a) and gamma (rIFN-gamma) interferons on three different treatment schedules. On treatment schedule A, 13 patients received i.m. rIFN-alpha-2a and rIFN-gamma simultaneously at a 1:1 ratio by dose units (2 X 10(6) U/m2), equivalent to a 1:10 ratio by protein weight. Results included severe constitutional symptoms in 62% of the patients and no partial remissions (PR) or complete remissions among 10 evaluable patients. On treatment schedule B, 25 patients received an i.m. injection of rIFN-alpha-2a and rIFN-gamma simultaneously at a 1:1 ratio by protein weight (2 X 10(6) U/m2 and 2 X 10(5) U/m2, respectively). This schedule was well tolerated, allowing a 25-50% increment in over 75% of the patients. Four patients (16%) achieved PR. On treatment schedule C, 15 patients received alternating weekly therapy: first rIFN-gamma (5 X 10(6) U/m2 daily for 7 days) followed by rIFN-alpha-2a (10 X 10(6) U/m2 daily for 7 days). One of 13 evaluable patients in treatment schedule C achieved an 80% tumor reduction and was rendered free of disease after the primary tumor was resected. Toxicity was similar in nature to that of rIFN-alpha-2a or rIFN-gamma separately. The toxicity of treatment schedule A suggested additive toxic effects. No distinct synergistic antitumor effect was observed.

Based on the in vitro and in vivo data suggesting synergistic cytolysis by the combination of 5-fluorouracil and interferon-gamma against a variety of malignant cell lines including a human colon carcinoma cell line (HT-29), we initiated studies in patients with advanced colon or rectal carcinoma. Forty-six patients received 5-fluorouracil as an intravenous injection on days 1-5 and recombinant human interferon-gamma as an intramuscular injection on days 1-14, followed by a rest period of 14 days; courses were repeated every 28 days. In the phase I study, cohorts of two patients received a stepwise dose level increase to achieve the maximum tolerated dose (MTD), at which a total of six patients were studied. The dose levels constituting the MTD were as follows: 5-fluorouracil (500 g/m2/day) and recombinant gamma-interferon (0.5 mg/m2/day). Four patients achieved a partial response in the phase I study. In the phase II study, 30 patients received therapy at the MTD. Among 29 evaluable patients in the phase II study, two patients achieved a partial response. Common toxicities included malaise, fever, anorexia, nausea and vomiting, and diarrhea. Transient severe myelosuppression was common but did not result in significant morbidity. Our data suggest that the combination of 5-fluorouracil and recombinant gamma-interferon did not have the same antitumor effect in patients as it had in the preclinical experiments.

Recombinant human gamma-interferon and recombinant human tumor necrosis factor are two representatives of a new class of antineoplastic agents. In vitro studies have suggested synergistic cytotoxic activities when the agents are combined. We report a phase I study of these two agents when administered daily for 5 consecutive days every 2 weeks in patients with advanced gastrointestinal cancers. Toxicity resulting from these agents was significant with hyperbilirubinemia representing the dose-limiting toxicity. Significant, although transient, myelosuppression was also observed. The maximal tolerated doses were 150 micrograms/m2/day for 5 days for each agent. Suggestive antineoplastic activity in biliary and pancreatic cancer was observed. Phase II trials of this combination are currently in progress.