Laser differential light scattering bioassays for selected antitumor agents.

Bioassay methods for actinomycin D, 6-thioguanine, and adriamycin which utilize laser light scattering from suspensions of drug-sensitive bacteria have been developed. In 3 hours, serum and urine levels of the 3 drugs can be reproducibily measured. A sample volume of 0.1 ml may be assayed with a precision of +/- 10%. The linear response range for actinomycin D was 0.03 to 3.0 microgram/ml. For 6-thioguanine and adriamycin the linear responses range was 0.03 to 10.0 and 0.25 to 10.0 microgram/ml, respectively. Thetively. The present studies further confirm the general utility of differential light scattering as a rapid and inexpensive assay procedure of broad application.

Laser light scattering bioassay for 1-beta-D-arabinofuranosylcytosine (ARA-C, NSC-63878).

A bioassay method for the estimation of ara-C in biological samples which utilizes laser light scattering from suspensions of drug-sensitive bacteria has been developed. The species employed in the assay was Streptococcus faecium var. durans resistant to methotrexate and 6-mercaptopurine. In less than four hours, serum and urine levels of ara-C can be reproducibly measured using the method. A sample volume of 0.1 ml containing 30 ng/ml may be assayed with a precision of +/-10 percent. The present studies further confirm the utility of the method as a rapid and relatively inexpensive assay methodology of broad application. Time variations of drug serum levels and urinary excretion rates in dogs are compared via the differential light scattering (DLS) assay, standard disc diffusion assay, and radiosotopic assay. The results obtained by the various methods are in excellent agreement.

Distribution and metabolism of prednisone in mice, dogs, and monkeys.

A chromatographic procedure that allows measurement of [3H]prednisone and [3H]prednisolone in biologic fluids and tissues was developed and used to determine some of the biochemical properties of prednisone and its distribution and metabolism in mice, dogs, and monkeys. When prednisone was added to mouse serum in vitro at a concentration of 50 microgram/ml, approximately half of the drug became bound to serum proteins. Homogenates of mouse liver metabolized prednisone to prednisolone. Following ip or oral doses of prednisone to mice, serum levels of prednisone, prednisolone, and other metabolites were maximum at 15 minutes. 20-Dihydroprednisolone was identified as a metabolite in several biologic samples. For dogs and monkeys given in iv dose of prednisone, disappearance from the serum was biphasic with apparent half-lives of 15 and 82 minutes and 33 and 78 minutes respectively. The serum levels of prednisone and prednisolone in mice, dogs, and monkeys were not increased beyond expected levels by simultaneous administration of iv doses of vincristine. The prednisone content of the kidneys, liver, intestine, and bile of a monkey killed 30 minutes after an iv dose was higher than that in serum. Oral doses of prednisone were absorbed erratically by dogs and monkeys. Only small amounts of prednisone and prednisolone were excreted in the urine of dogs and monkeys given an iv or oral dose.

Physicochemical considerations and pharmacokinetic behavior in delivery of drugs to the central nervous system.

The blood-brain barrier is not as rigid or as formidable as once believed. When experimental tumors of the central nervous system (CNS) and tumors of non-CNS origin are implanted intracerebrally (ic), they will grow and kill the host. It was once thought that only a few select agents, such as the nitrosoureas, were capable of crossing the blood-brain barrier and inhibiting tumor growth. There are now considerable data available which indicate that a variety of agents such as cytosine arabinoside, methotrexate, 6-mercaptopurine, 5-fluorouracil, procarbazine, melphalan, and cyclophosphamide, as well as the nitrosoureas, may significantly influence the growth rate of experimentally implanted ic tumors. A number of physicochemical factors may influence the penetration of the blood-brain barrier. These include pKa values, log-P values, and molecular size. Brain-level measurements indicate that most drugs have some accessibility to the CNS. Maximal effects against sensitive, ic implanted tumors are attained through maximal scheduling. Effective drug combinations should be sought to further enhance antitumor effects in the CNS.

A model for the kinetics of distribution of actinomycin-D in the beagle dog.

A pharmacokinetic model is presented for the distribution of actinomycin-D in the beagle dog. A simple, flow-limited model provides good simulations of the data at doses of 0.6 mg/m2 (0.03 mg/kg) and 2.7 mg/m2 (0.135 mg/kg) for most normal tissues. This implies that uptake of actinomycin-D in vivo is limited by tissue blood flow rate rather than by cell permeability. However, uptake by the testes is restricted by a blood-testis barrier, and a linear membrane-limited model is required to simulate the testis data. Linear binding of actinomycin-D to tissue is suggested by the fact that tissue concentrations are proportional to dose at least up to the lethal dose in dogs. The binding is also rapid and reversible as indicated by the tissue concentration curves which are parallel to the time course of the declining plasma curves.

A method for the measurement of L-phenylalanine mustard in the mouse and dog by high-pressure liquid chromatography.

The distribution of L-phenylalanine mustard (L-PAM) was studied in dogs and mice by high-pressure liquid chromatography. Separation of L-PAM from its products of hydrolysis was accomplished with a mu-Bondapak C18 column, a solvent system composed of 2-methoxyethanol/0.1% acetic acid, and solvent programming with a step gradient. Complete separation was effected in less than 15 min. The half-life for disappearance of L-PAM from mouse blood was 41 min, whereas that from dog blood was 29 min. The monohydroxy derivative of L-PAM, L-MOH, disappeared from dog serum with a half-life of 32 min. L-MOH was not detectable in mouse tissue other than blood at times greater than 15 min after injection. In the dog at 4 hr after injection, the tissue/serum concentration ratios were greater than 1 for liver, spleen, intestine, skeletal muscle, urinary bladder and gallbladder. The concentration of L-PAM in the bile was approximately 500 times higher than that in serum.

Distribution of tetrahydrouridine in experimental animals.

2-14C-tetrahydrouridine was prepared and used to determine the serum levels and excretion of tetrahydrouridine by varous experimental animals. In mice injected ip with the agent (50 mg/kg), the serum level of tetrahydrouridine was maximum (76 mug/ml) at 15 minutes. For rats injected with the same dose, the tetrahydrouridine content of serum was greatest (58 mug/ml) at 30 minutes. Within 3 hours, the serum content of the drug in both mice and rats fell to less than 10% of the maximum. The kidneys of mice selectively accumulated tetrahydrouridine; the concentration rose to 275 mug/g at 1 hour after injection. Nearly all of the dose was excreted unchanged in the urine of mice and rats in 24 hours. For a dog and a monkey given an iv dose (50 mg/kg) of tetrahydrouridine, serum levels of the agent were 210 and 200 mug/ml, respectively, at 5 minutes. The apparent half-lives for the initial phase of disappearance were 18 and 20 minutes and those for the later phase were 65 and 70 minutes, respectively. In 24 hours the dog and monkey excreted most of the dose as unchanged tetrahydrouridine. No metabolites were detected in the biologic samples from either species.

Disposition of [3H]actinomycin D in tumor-bearing mice.

A single, nonlethal dose of actinomycin D will cause total regression and cure of Ridgway osteogenic sarcoma in mice. A cure is not obtained with a single dose daily for 7 days, a dose regimen which kills 10% of normal C57BL/6 X DBA/2 mice. This suggests that actinomycin D is more effective on a single high-dose schedule than on chronic daily therapy. Analysis of drug exposure in Ridgway osteogenic sarcoma and normal mouse tissues following the single and multiple-dose regimens suggests the difference in therapeutic response is due to drug exposure at a higher concentration in Ridgway osteogenic sarcoma after the single high dose than after the multiple-dose regimen. This may be related to the higher drug concentration attained in blood following the single-dose regimen than is attained with the multidose regimen.

Laser differential light-scattering bioassay for methotrexate (NSC-740).

A new bioassay method utilizing laser light scattering from suspensions of drug-sensitive bacteria has been developed for the estimation of antitumor drugs in biologic samples. Changes in the light-scattering patterns of antibiotic-treated bacteria have recently been shown to provide a rapid and accurate indication of antibiotic sensitivity. Similar considerations for several antitumor drugs have shown the method capable of assaying 0.1 ml with drug concentrations as low as a few nanograms of drug per milliliter of sample. The first successful application of the methodology is described for the antitumor agent methotrexate. Studies of both drug-treated human serum specimens and dog serum levels and urinary excretion as a function of time indicate that assay results are available within 3 hours of preparing the serum. Time variations of drug serum levels and urinary excretion rates are compared via laser differential light-scattering assay, standard disc-diffusion assay, and previously published radioisotopic assays.

Tissue disposition of 3H-actinomycin D (NSC-3053) in the rat, monkey, and dog.

Tissue concentration of 3H-actinomycin D have been determined in the rat, monkey, and dog after an iv dose of 0.6 mg/m2 body surface area. The drug-tissue half-life was determined for various tissues of the rat, monkey, and dog. A mean drug-tissue half-life of 47 hours was calculated for the tissues of the dog. Exceptions were the testis and brain. Significant concentrations of 3H-actinomycin D failed to accumulate in the brain. Although testis drug concentrations were lower than most other tissues evaluated, the drug-tissue half-life was significantly greater than that of other tissues. In all species studied 3H-actinomycin D was rapidly depleted from serum after iv dosage, with concomitant accumulation of drug in the tissues. 3H-actinomycin D was excreted via the biliary and urinary routes in all species studied. No metabolites of 3H-actinomycin D were detected in the bile or urine of the rat, monkey, or dog with the methods employed. When expressed on a body weight basis, body surface area doses were more than threefold greater in the rat than in the dog. However, the average ratio of concentration X time values (rat C X T/dog C X T) for 11 different tissues of the rat and dog was only 1.3. The results strongly suggest that an equivalent dose of actinomycin D, with dosage based on a body surface area basis, results in nearly equal tissue-drug exposure for most tissues in various mammalian species.

Kinase and deaminase activity in a variety of subcutaneous mouse tumors.

Extracts of solid mouse tumors were examined for deoxycytidine kinase and deaminase activities. 1beta-D-Arabinofuranosylcytosine nucleotide was formed at a rate of 45 nmoles/hr by Glioma 26/57 and only 14 nmoles/hr by Ridgway osteogenic sarcoma. Deaminase activity was highest in Lewis lung (114 nmoles of 1-Beta-D-arabinofurano-syluridine formed per hr) and in CaD2 (104 nmoles of u-beta-D-arabinofuranosyluridine formed per hr). Deaminase activity in tumor extracts is sensitive to freezing, while deaminase activity in monkey serum is not. It was observed that kinase activity varies by as much as 50% in different cell lines of the same tumor. In the presence of tetrahydrouridine, kinase activity was significantly increased in most of the tumors studied.

Inhibition of deamination of 14C-cytosine arabinoside (NSC-63878): a useful biologic assay for tetrahydrouridine (NSC-112907).

Inhibition of the deamination of 14C-cytosine arabinoside by two lots of tetrahydrouridine was studied in monkey serum. The average inhibition of deaminase activity was 78% for tetrahydrouridine lot AJ39 (1.0 muM) when the concentration of cytosine arabinoside ranged from 44.2 to 170.7 muM; under the same conditions tetrahydrouridine lot AJ22 inhibited deamination by an average of 68%. Apparent Ki values were 0.26 muM for AJ39 and 0.43 muM for AJ22. The assay may be used to check the relative biologic activity of various lots of tetrahydrouridine.

Influence of tetrahydrouridine on the phosphorylation of 1-beta-D-arabinofuranosyl-cytosine (ara-C) by enzymes from solid tumors in vitro.

The phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) was studied in cell-free extracts from a variety of solid mouse tumors, L1210 ascites and in normal liver and spleen. Two apparent Michaelis constants were observed for kinase activity in Lewis lung (Km1, 4.15 muM; Km2 58.1 muM), sarcoma 180 (Km1 6.66 muM; Km2 56.18 muM), adenocarcinoma 755 (Km1 4.34 muM; Km2 50.0 muM) and l1210 (Km1 29.41 muM; Km2 41.67 muM). The Km1 values generally ranged from 5 to 20 muM 3H-ara-C while the Km2 values ranged from 20 to 60 muM 3H-ara-C. Normal spleen (Km 47.6 muM), normal liver (Km 10.0 muM) and Ridgway osteogenic sarcoma (Km 31.2 muM) had single Km values. In the presence of tetrahydrouridine (H4U), the in vitro phosphorylation of ara-C was increased as much as 91% in cell-free extracts from adenocarcinoma 755; lesser increases were observed in other tumor extracts. At low substrate concentrations, the apparent Km decreased or did not change in the presence of H4U, while at higher substrate concentrations the apparent Km was increased or did not change in the presence of H4U. In the presence of H4U, Vmax for kinase activity increased most in those tumors possessing deaminase activity.

Unexpected beta adrenergic effects of the antitumor agent, cyclocytidine, on rat salivary glands.

In addition to its potent antileukemic properties, cyclocytidine has a sialogogue action that depends on stimulation of beta adrenergic ereceptors of salivary glands. Furthermore, when chronically administered (for 3 days), cyclocytidine caused enlargement of parotid and submaxillary glands and heart that resembled the hypertrophy caused by chronic isoproterenol administration. The salivas evoked by cyclocytidine also closely resembled those evoked by isoproterenol, and were extremely viscous, and high in K+, (121 plus or minus 5.6, for submaxillary, and 42 plus or minus 2.9, for parotid), low in flow rate (0.007 mg/min times mg) and parotid saliva contained high concentrations of amylase (805 plus or minus 33 mg/mg gland). Cyclocytidine also caused marked emptying of parotid gland amylase. The cyclocytidine-induced salivary flow and gland emptying of amylase were prevented for 90 min when propranolol (but not dibenzyline or atropine) was administered prior to injection of the cyclocytidine. In addition, when the superior cervical ganglion was acutely removed, administration of cyclocytidine elicited salivary flow from the denervated as well as the innervated glands. These findings suggest that cyclocytidine does not affect salivary glands through indirect central or ganglionic actions. Cyclocytidine action does not exclusively involve beta receptors, since even in the presence of propranolol, secretory flow was evident after 90 min but when dibenzyline was given with the propranolol, complete blockade of cyclocytidine-stimulated saliva was effected. The dominant effect is, however, a beta adrenergic one. The undesirable side effects of cyclocytidine (parotid pain, postural hypotension, and cardiac hypertrophy) probably stem chiefly from its beta adrenergic properties and might be eliminated (or at least modified) by administration of propranolol with the cyclocytidine.