Monitoring CP-93,393 reaction mixtures by flow-injection analysis-mass spectrometry.

CP-93,393 is a drug candidate at Pfizer. Flow-injection analysis-mass spectrometry (FIA-MS) was used to monitor reaction completion for CP-93,393 reaction mixtures. FIA-MS provides essentially instantaneous results, is relatively simple to operate, and is a universal system that can be used to monitor any reaction as long as the product has a molecular weight that differs from the molecular weights of the reactants. The mass spectrometer for these studies employed atmospheric pressure chemical ionization. Samples were introduced into the mass spectrometer with a flowing stream of solvent.

Sacrospinous suspension made easy.

An orthopedic instrument, the Shutt Suture Punch System, has allowed rapid (less than 1 minute) and safe introduction of the suspending suture through the sacrospinous ligament, especially in the morbidly obese patient. Residents-in-training have performed the sacrospinous vaginal suspension on 30 patients without complications. The remaining aspects of the operation are unchanged.

Identification and evaluation of volatile compounds of vacuum and modified atmosphere packaged beef strip loins.

Beef strip loins were packaged and stored for up to 28 days at 3°C in high-oxygen barrier film under vacuum and in 100% CO(2), 40% CO(2)/60% N(2) and 20% CO(2)/80% O(2). As storage progressed, loins packaged and stored in 20% CO(2)/80% O(2) developed strong off-odors. 1-hexene, methyl thiirane, ethyl acetate, benzene and 1-heptene were detected in these packaged loins beginning at 7 to 14 days of storage. With the exception of 1-hexene, these compounds were not consistently detected in loins stored in vacuum, in 100% CO(2), or in 40% CO(2)/60% N(2), and these packaged loins developed much less off-odor during storage than loins packaged and stored in 20% CO(2)/80% O(2). A large number of volatile compounds from the headspace of the packaged loins originated from the packaging material. Lactobacillus plantarum became the dominant flora on loins stored under vacuum and under 40% CO(2)/60% N(2) while Leuconostoc mesenteroides subsp. mesenteroides predominated in loins stored in 100% CO(2). Pseudomonas putida eventually dominated on loins stored in 20% CO(2)/80% O(2).

Seasonal variation of lipids in femoral gland secretions of male green iguanas (Iguana iguana).

Femoral gland secretions were collected from 21 captive adult male green iguanas (Iguana iguana) in Orotina, Costa Rica, and San Diego, California, during the breeding (November) and nonbreeding (March) seasons. Lipids were extracted with methylene chloride, weighed, separated by thin-layer chromatography, and analyzed by gas chromatography-mass spectrometry. Free and esterified C14-C26 fatty acids, 5ß-cholestan-3α-ol (epico-prostanol), cholest-5-en-3ß-ol (cholesterol), cholest-5-en-24-methyl-3ß-ol (campesterol), cholesta-5,22-dien-24b-ethyl-3ß-ol (stigmasterol), cholesta-8,24(5α)-dien-4,4,14α-trimethyl-3ß-ol (lanosterol), cholest-5-en-24-ethyl-3ß-ol (ß-sitosterol), and two uncharacterized sterols were indicated. More lipids were recovered from femoral gland secretions obtained during the breeding than the nonbreeding months, indicating that secretion deposits may be more detectable during the mating season.

Identification of major lipids from the scent gland secretions of Dumeril's ground boa (Acrantophis dumerili Jan) by gas chromatography-mass spectrometry.

The scent gland secretions of Dumeril's ground boa (Acrantophis dumerili), pooled from two adult males and a female, were analyzed by gas chromatography-mass spectrometry. 2-Hydroxy-propanoic acid, hexadecanoic acid, cis-9-octadecenoic acid, octadecanoic acid, cholesterol, and 5-cholesten-3-one were indicated. These results are compared with those obtained in analyses of the scent gland secretions of other snakes.

Phase I and pharmacologic study of 72-hour infused 5-fluorouracil in man.

The relationships between the administered dose, clearance, and the toxicity spectrum of 5-fluorouracil (5-FU) administered as 72-hour constant infusion have been studied in 21 patients with advanced cancer. This was done as a pilot study for possible future combination using 5-FU as a radiosensitizer. Individual patients tolerated up to 65 mg/kg/24 hours, but serious toxicity appeared once as low as 35 mg/kg. Limiting toxicity proved to be "mixed" with upper intestinal symptoms (nausea and vomiting), stomatitis, and central nervous system signs all occurring in various patients. Central nervous system effects (both cerebellar and vomiting) proved as troublesome as stomatitis. There was only a general link between the administered dose and the subsequent toxicity grade, but a reasonably quantitative relationship emerged when the serum 5-Fu levels obtained and the degree of patient toxicity were compared. The clearance of 5-FU was confirmed to be nonlinear over the entire dose range studied (25-65 mg/kg/24 hours), consistent with a two-compartment model of drug metabolism. One compartment appears to be systemic (extra-hepatic) metabolism (probably anabolic removal) which is saturated at just below 15 mg/kg/day. Doses above that level lead to drug accumulation. No steady state was reached, contrary to previous reports. At the higher infusion rates, clearance progressively approaches that predicted by the assumption that the second compartment is splanchnic blood flow and catabolism. While 5-FU can be administered as a 72-hour infusion as one possible schedule for use as a single agent or for combined modality studies, CNS effects are quite troublesome in comparison to longer infusions to toxicity.

Relevance of the pharmacology of oral tegafur to its use as a 5-FU pro-drug.

We have studied the pharmacology of iv and oral tegafur (FT) and compared the results with similar studies using continuously infused 5-FU. All patients received daily abdominal irradiation as well as FT. In eight patients receiving 1.0 g/m2 of iv FT, serum FT levels were essentially the same as those in five patients receiving the same dose of oral FT. Oral FT appeared well-absorbed, even with abdominal irradiation. The mean serum FT achieved on a daily basis was a linear function of the oral FT dose between 1.0 and 2.5 g/m2 and was consistently about 1000-fold higher than the resultant 5-FU level. The major FT metabolite, dehydro-FT (DHFT), was persistently present at about ten times the 5-FU level. Because of their long half-lives, both FT and DHFT accumulate during continuous therapy. When the mean serum 5-FU levels with oral FT were compared to those found during continuous 5-FU infusions, we found that oral FT was the equivalent of low-level 5-FU infusion. Oral FT at 1.0 g/m2 was the equivalent of about 11.5 mg/kg/24 hours of 5-FU, increasing to about 17.5 mg/kg of 5-FU for oral FT at 2.5 g/m2. The pharmacologic properties of FT appear to dictate its most useful schedule (continuous oral dosing in multiple doses) and explain why FT alone is not ideal as a 5-FU pro-drug. In addition, insight into the pharmacokinetic limitations of FT also suggests means by which its usage may be improved, including its potential application as a radiosensitizer.

Phase I and pharmacologic study of 72-hour infused 5-fluorouracil and hyperfractionated cyclical radiation.

We have studied 21 patients infused for 72 hours with 5-Fluorouracil (5-FU) at progressive doses combined with hyperfractionated radiation. The schedule was chosen as being one capable of inducing 5-FU radiosensitization (RS). All patients were started at a daily 5-FU dose of 40 mg/kg/24 hours; doses were then escalated with each subsequent treatment cycle to limiting toxicity or until taken off study. Patients received between one and six infusion cycles. Every treatment cycle included coincident hyperfractionated radiation to various body areas including the abdomen, chest, and head and neck region. Radiation fractionation was invariant; 1,000 rad were delivered in four equal fractions. Two fractions of 250 rad each were given on days 1 and 2 of each three day 5-FU cycle, i.e. at approximately 0, 8, 24, and 32 hours into the drug infusion. Patients were followed for toxicity; serum 5-FU concentrations were determined using a high pressure liquid chromatographic assay. 5-FU clearances were calculated from the mean serum drug levels and the infused drug dose. The toxicity spectrum was not found to be significantly different from infused drug alone in this dose range save when the head and neck region received coincident irradiation. In that region the two anticipated toxicities combined in what appears to be a synergistic fashion to enhance mucositis. Most toxicities including gastrointestinal and bone marrow appeared dependent on the mean serum 5-FU level as did mucositis itself. 5-FU clearance was found to be non-linear in this dose region but did not appear influenced by radiation to any part of the body. This study shows that 72-hour infused 5-FU can be combined with external beam radiation and will produce reasonably predictable toxicity patterns which depend on the region of the body being irradiated. 5-FU toxicity correlates with mean serum drug level which is itself dependent on 5-FU clearance. Minor variations in 5-FU clearance therefore probably contribute to the natural range found in the dose-response relationship for infused 5-FU toxicities. Future studies should integrate this understanding of 5-FU pharmacokinetics into treatment regimens. The combination of infused 5-FU and coincident radiation appears useful in treating several tumor types, particularly squamous and squamous-like cancers. However, further scheduling and radiation fractionation studies are desirable to optimize 5-FU RS in man and to quantify late effects.

Phase I and pharmacologic study of oral ftorafur and X ray therapy in advanced gastrointestinal cancer.

We have treated 15 patients with advanced gastrointestinal carcinoma with a cyclical regimen of combined Ftorafur (N1-((2-furanidyl-))-5-Fluorouracil, a 5-FU pro-drug) and external beam radiation. The Ftorafur (FT) was administered orally in daily doses of between 1.0 and 2.5 g/m2/day in 3 divided doses in a Phase I format. The drug was given daily for 5 days along with conventional X ray treatment portals and daily radiation doses of 250 rad on each of the first 4 days of each treatment cycle. The patients were then rested for a minimum of 10 days or until all significant side effects had passed. The total number of 1,000 rad cycles and radiation dose were dictated by tolerance and by normal organ dose limitations. The most common toxicity in general, and the most common limiting toxicity was nausea and vomiting, in contrast to oral FT alone where diarrhea is more prominent. Stomatitis was seen only once and no other form of serious toxicity was encountered. Two-thirds of the patients responded in subjective terms (pain relief). There was 1 partial response to FT alone (pulmonary metastases outside the treatment field). The sole patient whose treatment field was outside the abdomen (chest portals for esophageal carcinoma) developed pneumonitis which contributed to his death. No other delayed effects were noted. Serum FT levels were related to the ingested dose and in the microgram range while serum 5-FU levels were in the nanogram range indicating slow decomposition of FT into 5-FU. The therapy was reasonably well tolerated at doses of 2.0 g/m2/day or lower with abdominal radiation. FT offers the potential for replacing intra-venous infused 5-FU as a clinical radiosensitizer.

Ionic strength dependence of the inhibition of acetylcholinesterase activity by Al3+.

Inhibition of acetylcholinesterase activity by Al3+ has been examined by initial velocity kinetics and by a first-order kinetic method. Both methods yield an inhibition constant of approx. 1.7 mM at 0.1 M ionic strength. The initial velocity study indicates a noncompetitive mechanism of inhibition by Al3+. Inhibition at 10 mM ionic strength shows a Ki of 0.03 mM. Evaluation of the ionic strength dependence concurs with the results of Nolte et al. (Biochemistry 19 (1980) 3705). An effective charge in the binding site of -9 predicts the ratio of inhibition constants at high and low ionic strength. Extrapolation to zero ionic strength gives a Ki0 = 0.34 microM.

Treatment with combined intra-arterial 5-FUdR infusion and whole-liver radiation for colon carcinoma metastatic to the liver. Preliminary results.

Twenty-eight patients with colon carcinoma metastatic to the liver were treated with continuously infused intra-arterial 5-fluorouracil deoxyriboside (5-FUdR) and cyclical whole-liver radiation (2000-3000 rad). Survivorship ranged from 25 days to almost 4 years and was a clear function of the extent of liver dysfunction at the time of initiation of this treatment. Difficulties in establishing the objective complete response rates in patients with minor imaging abnormalities were frequently noted. Both extracorporeal and permanently implanted arterial infusion devices have been employed, the results favoring the internal infusion units. Under ideal circumstances (early treatment, disease limited to the liver, and a permanent indwelling pump), a median survivorship of approximately 2 years can be projected with a significant number of patients rendered free of progressive cancer in the liver for months to years. The dose-limiting feature of this approach is treatment-related to hepatitis, which proved lethal in one of 28 patients thus far treated. Preclinical studies on the original and reduction of drug- and x-ray-induced liver toxicity should have high research priority.

Phase I and II trial of five-day infused 5-fluorouracil and radiation in advanced cancer of the head and neck.

Eighteen patients with advanced epithelial cancers of the head and neck region were studied for their tolerance and response to combined cycles of 120-hour infused 5-fluorouracil (5-FU) and external-beam radiation therapy. 5-FU infusions were given under conditions where radiosensitization would be expected at the higher infusion doses. Coincident radiation treatments were given as four sequential daily fractions of 250 rad each administered during days 1 through 4 of each five-day infusion cycle. The patients were rested for at least nine days after each cycle or longer until toxicity was resolved. The regimen was then repeated in each patient for a total of five treatment cycles. Thereafter therapy was consolidated, usually by boost radiation without drug. In sequential patient subsets the infusion load was progressively escalated in a phase I format. The complete response rate for stage IV patients was 75% with survival benefit compared to prior results. 5-FU dose-dependent combined modality loco-regional toxicity was demonstrated without significant enhancement of systemic toxicity of any form; 5-FU dose-dependent enhanced responsiveness and survival benefit is also suggested. Further scheduling and response studies of 5-FU under radiosensitizing conditions appear warranted.

Phase I-II study of 120-hour infused 5-FU and split-course radiation therapy in localized non-small cell lung cancer.

Thirty patients with locally advanced nonresectable non-small cell bronchogenic carcinoma were studied for tolerance and response to 120-hour continuously infused 5-FU (IFU) combined with chest x-ray therapy. The IFU was escalated in patient groups from 20 to 35 mg/kg/24 hours. Radiation was also escalated in patient subsets from 200 to 400 rads/fraction using a split-course technique to a total dose of 5000 rads. Tolerance was exceeded by IFU doses of 35 mg/kg/day. The complete response rate was ten of 28 patients (36%); a complete response was associated with a significantly prolonged survival. Radiosensitization of lung cancer by IFU appears clinically feasible in man.

Conservative management without alkylating agents of squamous cell anal cancer using cyclical 5-FU alone and x-ray therapy.

We have treated 11 patients with squamous cell anal carcinoma using a regimen of repeated cycles of 120-hour infused 5-FU (25 mg/kg/24 hours) and x-rays. Total radiation doses were between 3000 (palliative) and 4750 (curative) rads. Unlike all other equivalent combined-modality studies, no alkylating agent or drug other than 5-FU was used. With follow-up between 1 and 4 years, there has been only one local recurrence (coincident with terminal disseminated disease in a stage III patient treated palliatively). All other patients have maintained local control. Inclusion of alkylating agents (eg, mitomycin) in this approach can be questioned if the patient is willing to accept somewhat greater acute but reversible toxic effects.

Prognostic factors in the management of metastatic epidural spinal cord compression.

The results of 51 patients with metastatic spinal cord compression were analyzed. There were seven paralyzed patients, three received radiotherapy (RT) alone and four received laminectomy (L) + RT. No patient regained any motor function. Of six ambulatory patients, half received RT and half L + RT. All remained ambulatory after the treatment. Of 38 paraparetic patients, 20 underwent L + RT. Their complete, partial and nonresponse (CR, PR, NR respectively) rates were 25%, 60% and 15%, respectively. This result was clearly better than 18 other patients treated by RT alone of which only 22% regained ambulation (CR = 22%) while 67% were NR and 11% had a PR. In this series combined modality therapy appears better in paraparetic patients. Five patients with radiosensitive tumors all had CR/PR whether treated by RT or L + RT. Patients with epithelial tumors treated by L + RT had a PR (CR + PR) of 71% while RT alone gave only 25%. On the basis of this analysis we conclude: (1) ambulatory patients respond satisfactorily to RT alone; (2) paraparetic patients with radiosensitive tumors do well with RT alone while such patients with epithelial tumors merit L + RT; but (3) paraplegic patients rarely benefit from either modality; (4) pain control appears a useful measure of minimally adequate radiation dose in individual patients.

A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase.

A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H+ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88-95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of KI for edrophonium, decamethonium and Al3+.

Bleomycin, cyclophosphamide and radiotherapy in regionally advanced epidermoid carcinoma of the head and neck.

We have treated 24 patients with squamous carcinoma of the head and neck and advanced regional (N2-3) disease. The regimen consisted of 3 cycles, each of 28 days. Cyclophosphamide (1 gm/m2 I.V.) was given on day 1, bleomycin (15 u I.M.) on days 2, 4, 9 and 11, and ionizing radiation (60Co, 180 rad/fraction) days 1-5, and 8-12. No therapy was given on days 13-28. After three cycles of therapy, 13 patients had a complete response; following further therapy (surgery, interstitial or external beam radiation), 16 patients were free of disease. However, remissions were not durable and 11/16 patients recurred loco-regionally with a median time to recurrence of 5 months; most (7/11 also developed distant metastases. These patients have biologically aggressive disease and may have a worse prognosis than patients who are Stage IV based on a T4 primary lesion only.