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The efficiency of hemodialysis and controlled sequential ultradiffusion (CSU) for the elimination of toxic drug concentrations was tested by in vitro-investigations. In the 6 benzodiazepin derivatives tested, the clearance is markedly higher at a blood flow of 200 ml/min than at 100 ml/min. Pyrithyldione, glutethimide and phenobarbital are better dialysed than the benzodiazepines with exception of chlordiazepoxide. In comparison with hemodialysis, hemofiltration by means of CSU was less effective because of the small amount of ultrafiltrate obtained.

Ten milligrams of diazepam were injected intraamniotically in 8 mothers prior to therapeutic abortion between 12 and 19 weeks. The diazepam concentrations in the maternal plasma were comparable to those found after the same intramuscular diazepam dose to the mother. The concentration of diazepam in the amniotic fluid 12 to 18 hours after the injection was no longer significantly higher than in the maternal plasma. The concentrations of diazepam in the fetal plasma, liver and brain were comparable to the concentrations resulting from a 10 mg intramuscular diazepam dose to the mother about 2 hours before legal abortion. The feto-maternal ratio of diazepam was of same magnitude as after the intramuscular application to the mother. The results indicate that the disappearance of diazepam from the amniotic fluid in this stage of pregnancy occurs extraplacentally, through the mambranes into the uterine circulation. In the treatment of a fetus with drugs having properties similar to diazepam, intra-amniotic administration is no better than intramuscular administration to the mother.

The clinical effects of pentobarbital, 100 mg, and diazepam, 10 mg, given orally as preoperative medications before dental surgery were tested in a double-blind study in 50 adult patients. The concnetrations of pentobarbital and diazepam (plus its three active metabolites) in plasma, measured by gas chromatography, were correlated with their clinical effects as assessed both subjectively and objectively (sedation, apprehension, excitement, dizziness, pre- and post-operative emetic effect, increase or decrease in systolic blood pressure, pulse rate, and ease of venipuncture). No significant difference in the effects of these two agents was observed, nor was there any obvious relationship between the concentration in plasma and clinical effect.

The clinical effects of flunitrazepam and oxazepam as oral premedicants were tested in a double-blind study of 69 otorhinolaryngologic patients. Flunitrazepam had a somewhat higher sedative effect (p less than 0.10) and moderated the increase in systolic blood pressure significantly (p less than 0.005) more than did oxazepam, but as regards the other parameters tested no significant differences were found (sleep, apprehension, excitement, dizziness, emetic effect, headache, increase in heart rate, venepuncture). In some patients a profuse salivary secretion was observed despite intravenous injection of atropine just before the induction of anesthesia. Our results support earlier claims of flunitrazepam's relatively strong sedative and anxiolytic properties, but on the whole the difference in clinical effects of these benzodiazepine derivatives was not marked.

Midazolam given orally the night before and on the morning of operation had a distinct subjective pre-operative sedative effect as compared with placebo. Patients receiving midazolam also experienced less apprehension and excitement before surgery, but in relation to quality of sleep, the difference between the two groups was not statistically significant. Antidiuretic hormone (ADH) concentrations were determined just before induction of anaesthesia and were significantly lower in the midazolam group (2.14 pg/ml, SD 0.96) than in the placebo group (3.07 pg/ml, SD 1.73). Our results show that midazolam is a useful sedative anxiolytic oral premedicant, which appears to prevent initiation of a stress reaction before induction of anaesthesia.

The plasma protein binding of diazepam and of its main metabolite, N-demethyldiazepam, was measured in patients with renal disease and in healthy volunteers by ultracentrifugation. The total and non-protein bound concentrations of diazepam and N-demethyldiazepam, and the concentration of unconjugated oxazepam in the plasma were determined by gas chromatography after an oral 10 mg dose of diazepam. In the volunteers 98% of diazepam and N-demethyldiazepam were bound to the plasma proteins. In patients with renal disease the corresponding values were 92% and 95%, respectively. In the patients with renal disease no correlation could be found between the percent protein binding of diazepam or N-demethyldiazepam in the plasma and the serum creatinine concentration. Considerable variations in diazepam concentrations at 1 and 24 hours and in N-demethyldiazepam concentrations at 24 hours were found in the patients with renal disease. In contrast to the volunteers, 5 patients out of 28 with renal disease had measurable amounts of unconjugated oxazepam in the plasma. The deficient ability of the plasma proteins of patients with renal disease to bind diazepam may increase its clinical effect.

Plasma concentrations of nitrazepam were measured by gas-liquid chromatography in: young healthy volunteers, in geriatric and psychiatric patients and in epileptic children. The disposition of nitrazepam was described in terms of a two-compartment open model. After a single oral dose of nitrazepam 5 mg the most prominent differences between the experimental groups were in the beta-phase half-life mean 29 h in the young volunteers and 40 h in geriatric patients , and in the apparent volume of distribution during the beta-phase of 2.4 vs 4.8 1/kg. Total plasma clearance and the average steady state concentration in both groups were equal. The plasma level rose at a rate proportional to the beta-phase half-life, and so, they were achieved more rapidly in the young than in the old subjects (3.5 vs 7.5 d). No change in steady-state level or in the half-life of nitrazepam were found during long term treatment, which indicates lack of enzyme induction or inhibition. In 95% of the epileptic children with a good to fair clinical response, the plasma concentration of nitrazepam was 40-180 ng/ml (mean 114 ng/ml). As all of the patients were on combined antiepileptic therapy, no attempt was made to correlate plasma level with therapeutic response.

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.

Subrenal capsule assay (SRCA) is a promising method in cancer research and in the selection of individual chemotherapy for cancer patients. The laboratory procedures of SRCA are as follows: on day zero 1 X 1 X 1 mm pieces of fresh human tumour are carefully prepared. The pieces are transferred by a trocar under the outer capsule of normal immunocompetent mice, one piece to each. The mice (about 30-40/test) are divided randomly into groups of at least five. The size of each piece (initial size) is measured in situ by a stereomicroscope fitted with an ocular micrometer. On days 1-5 cytostatics are administered to the animals. On day 6 the animals are killed and the final tumour size is measured. The difference between the final and initial tumour size describes the response of the tumour to the treatments. The critical point of the assay is the selection of the tumour fragment which has to be representative, as homogeneous as possible and contain living tumour cells. When the sample is in pieces of about 3 X 3 mm (4-5 tumour fragments are sufficient for the assay) in the culture medium, it may be stored in an ice bath or at room temperature for one day. During this time, though as rapidly as possible, the sample has to be delivered to a research laboratory where the SRCA is carried out.

From 1982 through 1986 the subrenal capsule assay (SRCA) was used to test the sensitivity of 304 gynecological malignant tumors to cytotoxic drug combinations. Most of the tumors were ovarian cancers. Ninety-five percent of the assays were evaluable. Histological and ultrastructural studies were performed on transplants derived from ovarian cancers. Besides cytotoxic drugs, the activity of interferons was also studied by SRCA. Clinical comparisons were available in 43 patients with advanced disease; the predictive accuracy rate of the assay was 77%. The histological, ultrastructural and clinical studies indicated that SRCA is a reliable predictive test, which aids in the selection of proper therapy for individual patients.

Rous sarcoma cells were implanted into the kidney of rats. After 5 days of growth the renal tumor was used for comparing histology with glucose 6-phosphatase (G6Pase) enzyme histochemistry (EHC) and 18F-fluoro-2-deoxyglucose (FDG) auroradiography (ARG). It was found that the regions of the kidney tumor that had retained normal kidney structures were devoid of FDG, whereas there was histochemical staining of normal cortical areas. Regions of tumor growth, on the other hand, retained FDG and lacked G6Pase. Necrotic areas did not accumulate FDG. There was a dramatic decrease in the areas of G6Pase activity as a result of tumor infiltration in the kidney. The results show that FDG, currently being evaluated as a tumor detecting radiopharmaceutical indeed accumulates into areas of vital malignant growth, and they indicate that FDG positron emission tomographic (PET) images reveal the true anatomic location of malignant tissue.

Six women, 13 to 16 weeks pregnant, and 12 women at 38 to 40 weeks gestation, received oral oxazepam about 12 h before legal abortion, by hysterotomy in the former and before elective caesarean section in the latter group. The concentrations of free and conjugated oxazepam in maternal and fetal plasma were determined by gas-liquid chromatography. In early pregnancy the mean ratio between the plasma concentration of total (free + conjugated) drug in the umbilical cord and a maternal vein was 0.6 whereas in late preganancy the ratio vein was 1.1. Both in early and late pregnancy, the free and glucuronide conjugate of oxazepam were found in the fetus at concentrations which indicated transplacental passage of the parent drug and its metabolite. There was great interindividual variation in the plasma levels both of free and conjugated oxazepam.

Rat Walker 256 carcinosarcoma, human MCF-7 cell line and a specimen of ovarian serous cystadenocarcinoma were cultured in vitro and exposed to different cytostatic drugs. The drug effects were evaluated by bioluminescence, i.e., by measuring the levels of adenosine triphosphate (ATP), the basic energy source of the living cells. The intracellular ATP was released by TCA or NRS -reagent, and the ATP levels were measured directly from an aliquot of the growth medium without any extraction or precipitation steps. ATP level was significantly correlated with cell number, viability, [3H]-thymidine incorporation and stem cell assay. The most important advantages of bioluminescence method was speed, technical simplicity and good sensitivity (about 500 cells/sample easily quantitated, the results are seen directly within a few seconds) and flexibility (any cell line and drug may be studied by many different test designs). ATP method obviously describes the "well- being" of the cultured cells. According to our experience, the ATP-bioluminescence method is a powerful alternative to any other cell growth estimation method in vitro. It can be used especially in primary screening of the cytostatic activity of any known or unknown substance as well as in attempts to select an individual drug therapy for patients with cancer.

The combined and separate action of the antiestrogen toremifene (TOR) and recombinant rat gamma interferon (RIF) was studied in rat mammary cancer induced by dimethylbenzanthracene (DMBA). The content of ATP and 14C-fluorodeoxyglucose (FDG) was also determined from fine needle aspiration biopsies (FNAB). RIF alone had no antitumor activity, when measured as the average number of new tumors appearing in RIF and control animals (2.4 vs 2.4 new tumors per animal), while TOR and TOR + RIF had a significant effect (1.2, P less than 0.05 and 0.6, P less than 0.01). Morphometrically, there was a significant decrease in the amount of epithelium in the tumors of the RIF + TOR animals (65% vs 82% in the controls, P less than 0.05); there was conversely an increase in the stromal component (25% vs 14%, NS). It appears that an increase of the stromal compartment is part of the healing process. The feasibility of the FNAB-technique was shown by the finding that there was a close correlation between FDG and ATP content in almost all the groups before and after treatment. Thus, FDG and ATP measure the same phenomenon, i.e., energy content. There was a large variation in the contents of ATP and FDG within and among the groups, which invalidated considerations regarding the predictive value of ATP and FDG content in tumors subject to treatment.

Great interindividual variation was found in response to flunitrazepam when the latter was used as an induction agent for general anaesthesia in elderly, poor-risk patients. However, no significant changes in the pharmacokinetics of this nitrobenzodiazepine derivative were found, which suggests that there are pharmacodynamic alterations in response to the drug with advancing age. A sudden but transient drop in blood pressure was found in three out of 12 patients, even although flunitrazepam was given, i.v., in low 0.3-0.5 mg incremental doses. A marked amnesic effect was found. No analgesics were needed in the recovery room (2 h), supporting the evidence that flunitrazepam has an analgesic-sparing effect. Flunitrazepam resulted, in general, in smooth induction of anaesthesia, but a long time was needed for induction.

By random allocation 41 patients received 1 mg flunitrazepam orally the night before operation and 1 mg on the morning of operation (group 1), and another 41 received 100 mg pentobarbital orally the night before operation, followed by intramuscular scopolamine (0.006 mg/kg) + morphine (0.02 mg/kg) on the morning of operation (group 2). All patients received 0.5 mg atropine intravenously just before the induction of anesthesia. The patients in group 2 were better sedated and had less salivary secretion than those in group 1, but otherwise both were comparable. In group 2 the induction requirements of thiopentone were significantly decreased in comparison with group 1, again indicating a more potent sedative effect. Because even in the total scoring of the results there was no significant difference between the two groups, the easy oral route of administration of flunitrazepam offers a clinically relevant alternative to the conventional premedication. In some of these E.N.T. patients who received flunitrazepam, intravenous atropine given just before the induction of anesthesia was unable to prevent salivary secretion. Oral benzodiazepine derivatives (flunitrazepam) appear to be useful before surgery as the old type of premedication (oral pentobarbital + i.m. scopolamine and morphine).

Free and total concentrations of amikacin, tobramycin and gentamycin were measured separately in the rat kidney after equal weight by weight doses. The accumulation of aminoglycosides followed the order amikacin less than tobramycin less than gentamycin. The ratio between free and total aminoglycosides was similar (about 0.6) in all 3 aminoglycosides and independent on the length of administration.

Thirty-four patients were allocated at random to treatment with 1 mg of flunitrazepam, orally, the night before operation, and 1 mg on the morning of operation (Group 1), and another 34 to treatment with 100 mg of pentobarbital, orally, the night before operation, followed by intramuscular atropine (0.01 mg/kg)+pethidine (meperidine 1 mg/kg) on the morning of operation (Group 2). The patients in both groups slept equally well. As far as apprehension and excitement (= anxiolytic effect) just before induction of anaesthesia were concerned, oral flunitrazepam proved to be markedly better than i.m. atropine+pethidine. There were no significant differences in cardiovascular variables between the two groups. From the anaesthesiologist's point of view, atropine had beneficial antisecretory effects, but, from the patients point of view, it caused only a subjective unwanted effect (dry mouth). In our opinion, oral flunitrazepam is a useful alternative agent for routine premedication. However, when used without i.m. atropine, excessive salivary secretion in some patients may occur and be disturbing, especially during extubation.

In a double-blind randomized study 40 patients received 1 mg flunitrazepam, 40 patients received 10 mg medazepam, and 40 patients received placebo p.o. the night before surgery. On the morning of surgery each patient received another identical oral dose of the product or placebo taken the previous night. On the average, the flunitrazepam group slept better, was better sedated, and was less anxious than the medazepam or placebo groups. Similarly, as a reflection of diminished autonomic reactions, the patients receiving flunitrazepam had fewer cardiovascular changes. Flunitrazepam significantly decreased the amounts of thiopentone needed for induction of anesthesia. Medazepam did not. The pronounced sedative, sleep-inducing, and anxiolytic effects of flunitrazepam appear to be of great clinical importance for its use in anesthesiology. Repeated administrations of medazepam seem to be required to produce an evident clinical effect of the drug, possibly via the slow accumulation of metabolites. The main difference between these two benzodiazepine derivatives as regards clinical response therefore seems to be pharmacokinetic in origin.

The authors studied the effect of indomethacin and naproxen on the changes of renal prostaglandin E and F2 alpha concentration in experimental kidney infection, as well as the action of arginine-vasopressin in healthy rats. Naproxen proved to be an effective inhibitor of prostaglandin synthesis, as did indomethacin. In control animals an increased prostaglandin E and F2 alpha synthesis was observed caused by arginine vasopressin. It is supposed that ADH--depending on its concentration--has a metabolic modulator role in prostaglandin synthesis, which raises the possibility of a self-regulatory mechanism of water reabsorption.

Walker 256 carcinosarcoma was shown to be sensitive to mitomycin C in vitro. In order to make practical recommendations for the clinical intravesical use of mitomycin C, this cell line was used to evaluate the activity of mitomycin C under different conditions. Mitomycin C loses its antitumour activity rapidly at pH's below 6. At higher pH's (up to 10) mitomycin C is stable and suitable for intravesical application. To secure a sufficiently high pH in the bladder during intravesical treatment, phosphate buffer 0.05 M with a pH of 7.4 is recommended. Constituents of urine very little decrease the activity of buffered mitomycin C during a 2 h application. Prednisolone, which has been suggested to prevent the harmful chemical cystitis, has no inhibitory effect on the activity of mitomycin C.

The effects of pH on the antitumour activity of six cytostatics--cisplatin, doxorubicin, ethoglucid, mitomycin C, thiotepa and VM-26--used in intravesical instillations were tested in vitro. The activity of cytostatics was determined on the Walker 256 carcinosarcoma cell line by using ATP-bioluminescence method. Cytostatics were incubated at different pH's (5.0-7.4) for 2 and 4 h. The activity of most of the cytostatics was not affected by pH, but mitomycin C had somewhat lower activity at pH 5.0 at 4 h and cisplatin at pH 6-7 at 2 and 4 h. Because the pH of normal urine is within the range used in this test, the use of a buffer solution during the intravesical instillation therapy is not necessary. However, according to our clinical experience, buffering is recommended to avoid chemical cystitis.

The subrenal capsule assay (SRCA) was used to study the sensitivity of breast cancer to cytostatic drug combinations. The results were compared with steroid receptor status. Forty-five of 46 SRCAs (98%) were macroscopically evaluable. However, a histological study implied that the transplants should also be evaluated histologically, because in only 14/21 (67%) of the control SRCAs examined were histologically viable tumor cells seen. An inflammatory cell reaction was noticed in half of the cases. In the groups treated with cytostatics only 3/21 (14%) had vital tumor cells, whereas inflammation was evident in 4/21 (19%) of the cases. The rate of resistance to A + CTX was 30%. By testing several drug combinations against each tumor the rate of chemoresistance was reduced to 10%. The differences between A + CTX and the best combination were statistically significant (P less than 0.05). Of the tumors 79% were ER-positive and 67% PR-positive. Receptor-negative tumors tended to be more sensitive to cytostatics than receptor-positive tumors (100 vs 85%). The difference was not, however, statistically significant. It can be concluded that the SRCA under standardized conditions is a good method for studying the response of individual breast cancers to chemotherapy.

The uptake of various labeled compounds by tumors was studied by double-tracer whole-body autoradiography (DTWBA) in rats. Each animal carried two types of tumors: mammary carcinomas and the Walker 256 carcinosarcomas. The markers used were [18F]- and [3H]fluorodeoxyglucose (glucose utilization), [3H]thymidine (cell proliferation), [11C]methionine (amino acid metabolism) and [11C]- and [3H]toremifene (estrogen-receptor-avid agents). In each experiment, the distribution of a substance labeled with short-lived radionuclide (11C or 18F) was compared with that of another substance labeled with a long-lived nuclide (3H). Quantification was done by combining computerized image analysis of the autoradiograms with liquid scintillation counting of punched tissue pieces obtained from the cryosections. The relationships between the uptakes of the various radiopharmaceuticals were recorded in tumors and normal tissues. The dynamics of [18F]fluorodeoxyglucose and [11C]methionine were determined in tumors and some selected tissues by positron emission tomography (PET). The uptake rate between fluorodeoxyglucose and thymidine in the mammary tumor was five times higher than the ratio in the Walker tumor. The corresponding figure for FDG/methionine was four times. Thymidine, compared with methionine, was twice as efficient. Thus, the mammary tumors were best imaged with FDG or thymidine. The non-steroid antiestrogen toremifene was taken up in very low amounts by these tumors. By DTWBA, experimental tumors may serve as their own control.

Six women from 14 to 17 weeks pregnant, and 12 woman from 36 to 40 weeks pregnant, were given nitrazepam 5 mg orally about 12 h before legal abortion by hysterotomy in the former group and elective caesarean section in the latter group. The concentration of nitrazepam was determined by gas-liquid chromatography. Binding to plasma proteins was evaluated by separation of the protein-free fraction by ultracentrifugation. In the first group (early pregnancy) the level of nitrazepam was found to be lower in the fetal than in the maternal circulation. The concentration in amniotic fluid was still lower. In the latter group (late pregnancy) the concentration both of unbound and total nitrazepam in maternal and fetal plasma were in equilibrium, which indicated an increase in transplancental transfer in late pregnancy. The percentage of unbound nitrazepam in both cases was 12%.

The clinical effects of flunitrazepam and lorazepam as oral premedicants were tested in a double-blind study in 81 gynaecological patients. Flunitrazepam showed a higher sedative effect (p < 0.05), but in regard to other parameters tested, no significant differences were found (sleep, apprehension, excitement, dizziness, emetic effect, headache, increase or decrease in systolic blood pressure, increase in pulse rate, venipuncture). In 9 per cent of the patients treated with lorazepam, a prominent muscular relaxation with slurred speech was observed, but in none of the cases treated with flunitrazepam. Our results support the earlier claims of flunitrazepam's relatively specific sedative property, but on the whole the difference in the clinical effects of these benzodiazepine derivatives is not marked.

The 6-day subrenal capsule assay (SRCA) in normal immunocompetent mice was used to assess response of vulvar and cervical cancers to chemotherapy. Twenty-five out of 31 assays (81%) were evaluable. The previously treated tumors tended to be less sensitive than the untreated tumors (20 vs 33%). Three to five cytotoxic drugs or drug combinations were tested against each individual tumor. In the whole material the combination of cisplatin and etoposide (VP-16) was significantly more effective than bleomycin (P less than 0.01, chi 2 test). The combinations adriamycin + cyclophosphamide + cisplatin and cyclophosphamide + methotrexate were also rather effective. Preliminary clinical correlations were positive. The reliability of the SRCA was discussed, and it was concluded that the assay is a promising predictive method for individualizing chemotherapy.

The subrenal capsule assay (SRCA) as an in vivo predictive test is reviewed. The main topics include the technique, the evaluability, the reliability (both histological and clinical aspects) and the usefulness of the SRCA in fresh tumor samples.

Oestrogen receptor (ER) and progesterone receptor (PR) content and the response in vitro to tamoxifen (T), medroxyprogesterone acetate (MPA) and to a combination of the two hormones were determined in 21 epithelial ovarian carcinomas. The response was assessed by the level of adenosinetriphosphate in the cells. ER and PR were detected in 62% and 57%, respectively, with significant variations between the different histopathological cancer types. ER and PR predicted the response in vitro in 62% of the tumours exposed to the combined hormones, and in 38% and 33% of those exposed to T and MPA, respectively. The value of steroid-receptor determinations in selecting the proper hormonal treatment in ovarian cancer is significantly reduced because of the high proportion of incorrect predictions.

The subrenal capsule assay (SRCA) was used to test the sensitivity of gynaecological tumours to chemotherapy. The total number of the assays was 95, ovarian cancer being the most often tested. Of the SRCAs 92% were evaluable. Of the 59 evaluable assays for ovarian cancer, 52% showed sensitivity, 41% intermediate sensitivity, and 7% resistance to chemotherapy. The previously treated tumours were less sensitive than the untreated ones (p less than 0.01). To the most often tested drug combinations, DOX (doxorubicin) + C (cyclophosphamide) + DDP (cisplatin), DOX + C + Tegafur, and C + V (vincristine), resistance was observed in 10-20% of the assays. However, there was marked interindividual variation. Twenty-five assays of the squamous cell carcinomas were evaluable. Of the tumours 28% were sensitive, 48% intermediately sensitive, and 24% were resistant. Five of 7 sensitive tumours were previously untreated. The combination of DDP and VP-16 (etoposide) was tested most often, followed by Bleo (bleomycin) as a single agent. DDP + VP-16 was significantly superior to Bleo (p less than 0.01). The results of the SRCA and the clinical outcome of the patients could be compared in 31 cases. An overall predictive accuracy rate of 81% was achieved. It was concluded that the SRCA has predictive value in determining chemotherapy responsiveness of gynaecological tumours.

The chemotherapeutic potential of 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) was examined in vitro and in vivo. The compound was toxic in 2-day cultures; 7, 66 and 88% inhibition in the growth of L1210 cells was achieved with 1, 10 and 100 microM 5HmdUrd, respectively. The maximal plasma concentration of 5HmdUrd at 15 min after a single i.p. injection (100 mg/kg) in DBA/2 mice was 193-244 mumol./l and the compound had a logarithmic disappearance curve with a half-life of 20 min. Chemotherapy given as two daily i.p. injections of 5HmdUrd (100 mg/kg) for five successive days resulted in a 239% increase in median lifespan and 2/6 long-term survivals among DBA/2 mice bearing leukemia L1210. This treatment resulted in temporary neutropenia and thrombocytopenia, which were followed by rebound thrombocytosis and neutrophilia of short duration. Our data indicate that 5HmdUrd can successfully be used in experimental cancer chemotherapy in vivo.

The subrenal capsule assay (SRCA) in normal immunocompetent mice was performed from 1331 implants of 43 human colorectal carcinomas to evaluate the possible applications for clinical chemosensitivity testing. Also the effect of an immunosuppressive agent, cyclosporine, was tested on the growth of tumours. Histologically in all except one of 23 saline-treated tumours the original tumour tissue was replaced by granulation tissue and inflammatory cells. This was also true in cyclosporine-treated mice since in only one of the nine tests tumour cells were observed. The macroscopic growth of the implants in the cyclosporine-treated mice was significantly less than in the saline-treated mice. Flow cytometric DNA-analysis revealed that the difference between macroscopic growth of saline and cyclosporine-treated groups was observed only in DNA-diploid tumours. We conclude that new methods are required to preserve the viability of human colorectal carcinoma in the SRCA.

The transport system for organic acids in the kidney is not fully developed in the neonatal period. The effect of repeated administrations of ethacrynic acid on the renal excretion of p-aminohippurate (PAH) was studied in rats of different ages. Pretreatment with ethacrynic acid was followed by an increase in the renal excretion of PAH in 33-, 55-, 105- and 240-day-old rats but not in newborn rats. In 55-day-old rats the increase in renal excretion of PAH after pretreatment with ethacrynic acid was not associated with any consistent change of the glomerular filtration rate. It is concluded from these results that the stimulation of transport processes in the kidney by ethacrynic acid and some other drugs is linked with their affinity to tissue proteins.

In a single-blind randomized study 47 patients received 100 mg tofizopam orally as a premedication before minor surgery, 50 patients received placebo, an 50 patients no premedication. Both tofizopam and placebo significantly increased the subjective sedative effect of the patients, but there was no significant difference in any of the measured parameters between tofizopam and placebo. In another double-blind randomized study, 49 patients received 100 mg tofizopam three times orally before gynecologic operations and 49 patients received placebo. Compared with placebo, tofizopam had a stimulant action and decreased the excitement of the patients. The effect of tofizopam on apprehension + excitement was significantly better than that of placebo. According to these results, the active component seems to be an unknown metabolite of tofizopam causing a clear drug effect only after repeated oral doses of the parent drug. A slow accumulation in the central nervous system is also possible. In the drug response a wide interindividual variation was found.

Nitrazepam in plasma was determined by gas-liquid chromatography with a nickel-63 electron-capture detector, unchanged by a direct method and also by a hydrolysis method. The extraction in the direct method was carried out with benzenedichloromethane (90:10) and in the hydrolysis method with diethyl ether. The hydrolysis was performed with 6 N sulphuric acid. The hydrolysis product was extracted with toluene-n-heptane-ethyl acetate (80:20:5) directly from acid. Thus the commonly used change in pH was omitted. Nitrazepam concentrations in plasma were determined in 10 healthy volunteers after two oral doses (5 and 10 mg); 0.5 ml of plasma was used for each determination and clonazepam, methylbromazepam and methylnitrazepam were used as internal standards. The recoveries of the methods are almost quantitative (greater than 96%). The two methods are clinically comparable. The high sensitivity and specificity make these methods useful in clinical determinations of nitrazepam in plasma. Advantages and disadvantages of both methods are discussed.

Psychiatric patients (N = 26) were treated chronically (from 1 week to 12 years) with nitrazepam, because of insomnia. The patients gave their subjective estimations of the effects and side effects of nitrazepam. The concentrations of nitrazepam in the plasma were measured by 63Ni-EC-gas-liquid chromatography. The pharmacokinetics of nitrazepam were compared between the psychiatric patients and healthy volunteers (N = 11). The steady-state concentrations and the half-life of nitrazepam in the psychiatric patients were comparable to those of the healthy volunteers. The subjective hypnotic effect of nitrazepam was mostly good or satisfactory and remained unchanged during long-term treatment. Only a few, mild side effects were reported. Nitrazepam does not seem to cause enzyme induction with lowered plasma levels and may therefore be of special value in the treatment of chronic insomnia.

The in vitro protein binding of toremifene in human serum was measured by ultracentrifugation using 3H-toremifene together with unlabeled toremifene, 50, 500, and 5000 ng/ml. Of the total radioactivity 99.7 per cent was bound to the proteins independent of the concentration of the unlabeled drug. Binding of toremifene to different protein fractions was studied by adding 3H-toremifene and 500 ng/ml of cold toremifene to normal serum. The serum samples were exposed to agarose gel electrophoresis to fractionate different proteins. The radioactivity was localized using a position-sensitive proportional counter. After that the proteins were visualized by staining. Of the total protein bound radioactivity 92 per cent was bound to albumin, about 6 per cent to beta 1 globulin fraction and about 2 per cent to a fraction between albumin and alpha 1 globulins, part of this probably to alpha 1 acid glycoprotein.

The clinical effects of oxazepam and diazepam as oral premedicants were tested in a double-blind study of 60 children and 50 adults. The gas chromatographically measured concentrations of the active unconjugated forms of oxazepam and diazepam in the plasma were correlated to their clinical effects, as assessed both subjectively and objectively (sleep, sedation, apprehension, excitement, dizziness, emetic effect, headache, increase or decrease in systolic blood pressure, increase in pulse rate, venepuncture). No significant difference in the effects of these two benzodiazepine derivatives were observed, nor was there any obvious relationship between the plasma concentration and clinical effect.

The clinical significance of intramuscular premedication with 0.01 mg/kg of atropine in a procedure involving oral benzodiazepine premedication (15 mg midazolam the evening before surgery and on the morning of surgery) was investigated in a double-blind study. As far as sedation, apprehension, excitement, dizziness, emesis, and headache were concerned, there were no significant differences between group 1 (atropine) and group 2 (placebo) patients; however, both during and after anesthesia patients in group 1 had less excessive salivary secretion (especially during extubation). As a result of sympathetic overactivity, patients in group 1 had an increased heart rate and an increased incidence of supraventricular tachycardia. In group 1 intravenous infusion proved more difficult, and in addition, the patients complained more of subjective side effects (dry mouth). There was no significant correlation between the radioimmunologically measured serum concentrations and the clinical effects of atropine measured just before the induction of anesthesia. Substantial interindividual differences were found in these serum levels. From the anesthetist's viewpoint, atropine has both beneficial effects (antisecretory) and unwanted effects (cardiovascular effects). For the patient atropine caused only subjective unwanted effects. Midazolam, a new short-acting, sedative benzodiazepine derivatives, can be used without atropine as an oral premedicant.

Specimens from 16 freshly biopsied human tumors, two mammary adenocarcinomas, ten ovarian adenocarcinomas, two squamous cell carcinomas, one malignant histiocytoma and one chondrosarcoma of the bone, two human ovarian adenocarcinomas established by transplantation into nude mice and two adenocarcinomas induced in rat mammary gland were transplanted under the renal capsule of 510 normal immunocompetent mice and 180 rats and the effects of chemotherapy were evaluated. The results showed successful transplantation of all types of tumors in both animal species. Morphological analysis revealed preserved glandular structures with surface microvilli, mucin and CEA production and partially preserved basement membranes. Treatment with cyclophosphamide, vinblastine, adriamycin and cisplatin caused cell shrinkage, degradation and partial or total disappearance of the tumor cells. Vascularization was distinct in all specimens. A cellular infiltrate was found frequently but not consistently. A common end stage was a fibrotic scar with no cellular activity, occasionally giving a misleading impression of a growing tumor on gross observation. The results were obtained rapidly and suggest that the subrenal capsule assay would be useful for evaluating the sensitivity of human tumors to therapeutic manipulation, but needs supplementary histological examination.

The subrenal capsule assay in normal immunocompetent mice was used to test the responsiveness of ovarian cancer to combination chemotherapy. Of the assays, 42 were of untreated tumors and 19 of previously treated tumors. Fifty-nine (97%) of the assays were evaluable. The previously treated tumors were less sensitive than the untreated ones. Of the treated tumors 44% were sensitive, 33% intermediately sensitive, and 22% resistant versus 56, 44, and 0%, respectively, of the untreated tumors (P less than .01). Repeat assays for the tumors of seven patients were performed successfully after five to eight courses of therapy with the combination of doxorubicin, cyclophosphamide, and cisplatin. The responsiveness to this combination had weakened significantly (P less than .01); the response of only one tumor remained unchanged. The rates of resistance to the drug combinations doxorubicin-cyclophosphamide-cisplatin, doxorubicin-cyclophosphamide-tegafur, and cyclophosphamide-vincristine were 11, 10, and 21%, respectively; there was, however, considerable interindividual variation in tumor responses to these combinations. Of other combinations, hexamethylmelamine combined with 4-epidoxorubicin, aclarubicin, or chlorambucil and cisplatin had effect, whereas the combinations of cisplatin and etoposide and of tegafur and methotrexate or mitomycin were quite ineffective, as measured by the assay. The reliability of the subrenal capsule assay in normal immunocompetent mice is discussed, and it is concluded that the assay can be used to assess the response of ovarian cancer to chemotherapy, including multidrug therapy, without routine histologic control.

The toxicity and metabolism of a thymidine analogue, 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) were studied with human leukemia cells (HL-60) and with human platelets. 3 X 10(-5) M 5HmdUrd caused a 50% inhibition in the proliferation of HL-60 cells. The compound was hydrolyzed to 5-hydroxymethyluracil (5HmUra) by the enzyme thymidine phosphorylase (EC 2.4.2.4) present in leukemia cells; this catabolic product was non-toxic. The catabolism of 5HmdUrd by human platelet thymidine phosphorylase could be inhibited by 6-aminothymine. The toxicity of 5HmdUrd was effectively reversed by deoxycytidine and 5HmdUrd increased the incorporation of deoxycytidine into dCTP and DNA several fold. The two latter phenomena are explicable in terms of a feedback action to ribonucleotide reductase, resulting in deoxycytidylate starvation, which is a known effect of excess thymidine. We report here also our preliminary observations that 5HmdUrd is active against mouse leukemia in vivo.

The sensitivity to medroxyprogesterone acetate (MPA) and tamoxifen (T) and their combination was assayed in 13 patients with untreated endometrial cancer by an in vitro ATP-bioluminescence method. The method measures the levels of adenosinetriphosphate (ATP), the basic energy source of living cells. A tumor was considered to respond to the drug, if the proportion of living cells after manipulation was 50% or less from unmanipulated control culture. Estrogen (ER) and progesterone (PR) receptors were assayed by the DCC-method and the results calculated by Scatchard-analysis. ER (greater than or equal to 3 fmol/kg cytosol protein) was present in all tumors and PR (greater than or equal to 10 fmol/kg cytosol protein) in 85% of the tumors. The response rate in vitro to MPA was 67% (8 out of 12 tumors), to T 18% (2 out of 11) and to their combination 69% (9 out of 13). The G1 tumors responded statistically significantly better to MPA (p less than 0.01) and MPA + T (p less than 0.02) as compared to T. MPA produced higher cell kill of G1 than G2 tumors (p less than 0.05). The ER content correlated with the effect of MPA in vitro in 67%, with the effect of T in 18% and with that of their combination in 69% of the tumors. The PR content correlated with the effects of MPA in vitro in 83%, with the effect of T in 36% and with that of their combination in 54% of the tumors. It was concluded that the in vitro ATP-bioluminescence method provides valuable information besides steroid receptor determinations for sensitivity testing of endometrial cancer to hormones.

The concentrations of dapsone (DDS) and its acetylated derivatives (MADDS and DADDS) were determined in the serum and saliva after one oral dose of dapsone until 72 hr. The peak serum concentrations of DDS and MADDS were reached, on average, at 3.8--4.3 hr after the dosage. The amounts of DADDS were negligible. The elimination half-life of the first order kinetics was, on average, at 20--21 hr for both DDS and MADDS. The study group included 6 rapid acetylators and 4 slow acetylators with the mean ratios MADDS/DDS 1.0 and 0.19, respectively. No difference in the pharmacokinetics of DDS or MADDS could be seen between the rapid and slow acetylators. The protein-free fractions of DDS and MADDS were 50 and 41 per cent, respectively, of the total serum concentrations as measured at 8 and 32 hr after the dosage. The salivary concentration of DDS was, on average, 49 per cent of the total serum concentration during the whole study period. The salivary concentration of MADDS was 40 per cent, respectively. The elimination half-life of DDS and MADDS in saliva did not differ from that in serum. Between the salivary and serum protein-free concentrations a strict correlation existed (p less than 0.001). The salivary concentration of dapsone and its monoacetyl derivative reflect the protein-free, active drug in serum.