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PubMedDate not supplied

Development of an in vitro test battery for the estimation of acute human systemic toxicity: An outline of the EDIT project. Evaluation-guided Development of New In Vitro Test Batteries.

The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.

Biotransformation↗
PubMed1992

In vitro induction of CD8 expression on thymic pre-T cells. II. Characterization of CD3-CD4-CD8 alpha + cells generated in vitro by culturing CD25+CD3-CD4-CD8- thymocytes with T cell growth factor-beta and tumor necrosis factor-alpha.

We previously reported that CD25 (IL-2R p55)-positive CD3-CD4-CD8- murine thymocytes can be induced to express CD8 alpha (Lyt-2) by transforming growth factor-beta plus TNF-alpha in the presence of IL-7 (which is necessary to maintain the viability and differentiation capacity of CD25+CD3-CD4-CD8- thymocytes in vitro). The majority of cells recovered after 2 to 3 days from these cultures expressed CD8 alpha (but not CD3 or CD4). In this study, we have characterized these in vitro generated CD3-CD4-CD8 alpha + thymocytes and compared them with normal CD3-CD4-CD8+ thymocytes. Unlike normal CD3-CD4-CD8+ thymocytes that express CD8 alpha and CD8 beta (Lyt-3-chain) simultaneously, only a fraction of in vitro generated CD3-CD4-CD8 alpha + cells expressed CD8 beta. However, along with the induction of CD8 alpha and CD8 beta expression, the expression of other T cell differentiation markers (including CD2, CD25, and CD44) also changed in a manner corresponding to physiologic differentiation. Cell-surface phenotyping suggests that CD8 alpha + beta - cells are less mature than CD8 alpha + beta + cells. These in vitro generated CD3-CD4-CD8 alpha + thymocytes expanded and differentiated into the CD4+CD8+ stage as well as mature (CD3+) single positive (CD4+CD8-) and CD4-CD8+) stages in fetal thymus organ culture that had been depleted of lymphoid cells by treatment with 2-deoxyguanosine. The latter observation indicates that these in vitro generated CD3-CD4-CD8 alpha + thymocytes are responsive to other differentiation-inducing signals (including those that induce CD4) that exist in fetal thymus organ culture. These results suggest that in vitro generated CD3-CD4-CD8 alpha + thymocytes represent intermediate differentiation stages between CD25+CD3-CD4-CD8- and CD3-CD4-CD8+ cells found in normal thymus.

Animals↗
PubMed1994

In vitro drug testing of ovarian cancer using the human tumor colony-forming assay: comparison of in vitro response and clinical outcome.

The purpose of this study was to assess the prognostic value of in vitro drug chemosensitivity testing using the Hamburger-Salmon human tumor colony-forming assay (HTCA) in fresh tumor samples obtained from newly diagnosed patients with stage II-IV ovarian cancer undergoing maximum cytoreductive surgery and prior to platinum-based chemotherapy. The HTCA was performed on fresh ovarian cancers obtained from 93 patients at their initial exploratory laparotomy to evaluate in vitro sensitivity to cisplatin, carboplatin, and cyclophosphamide following a 1-hr drug exposure. Prospective clinical follow-up was performed on all patients with the primary study endpoints being pathologically proven complete response at second-look surgery and disease-free and overall survival durations. In vitro drug sensitivity was strongly dose-dependent. At a concentration of 5 micrograms/ml only 23% of tumor samples were sensitive (as defined by a > or = 50% decrease in tumor colony-forming units compared to controls) to cisplatin; 13% of tumors were sensitive to carboplatin at a concentration of 50 micrograms/ml and 11% to 4-OH-cyclophosphamide at a concentration of 1 microgram/ml. At doses which were 10 times the previously stated concentrations, the sensitivity rates to cisplatin, carboplatin, and 4-OH-cyclophosphamide increased to 72, 63, and 53%, respectively. Subjects were categorized as having drug-sensitive disease if HTCA results showed in vitro drug sensitivity to at least one of the agents used in their primary chemotherapy. Multivariate analysis failed to show any advantage in clinical response rate, progression-free interval, or survival duration for patients with drug-sensitive disease compared to drug-resistant disease; however, there was evidence of a trend toward an enhanced pathologically proven complete response rate in patients who had chemosensitive tumors in vitro. Second-look surgery was performed in 28 of 55 patients with optimal surgical resections and no clinical evidence of disease at the completion of their primary chemotherapy. Fifty percent (5/10) of patients with drug-sensitive disease achieved a pathologic complete response, while only 3/18 (17%) patients with drug-resistant tumors had a documented pathologic complete response (P = 0.13). As reported in other ovarian cancer studies, patient characteristics which were found to be significantly associated with survival were stage of disease (II-III vs IV), optimal primary surgical resection (i.e., < 1 cm2 residual tumor) vs suboptimal resection, clinical measurability of disease at initiation of chemotherapy, and response to primary chemotherapy. In conclusion, in vitro drug sensitivity, as measured by the HTCA, does not appear to be an independent prognostic factor for survival in patients with stage II-IV epithelial ovarian cancer who undergo standard treatment with tumor debulking surgery and primary platinum-based chemotherapy.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1993

An in vitro assay for drug sensitivity of Trypanosoma congolense using in vitro-derived metacyclic trypanosomes.

The sensitivity of seven populations of T. congolense to the salts of three trypanocides, diminazene, isometamidium and homidium, were determined in vitro using in vitro-derived metacyclic trypanosomes. The trypanosomes were incubated at 35 degrees C for 48 h with various drug concentrations (0.5 ng-50 micrograms/ml) and then transferred to cultures containing bovine endothelial-cell monolayers, to assess their viability over the following 5 days as compared to control trypanosomes that had been incubated without drug. The sensitivity to each drug was expressed as the minimum effective drug concentration which killed 100% of the trypanosomes in a given population within the 5 days. Using this assay, population IL 1180, characterised as being highly sensitive to all three drugs in vivo, required 10 ng/ml isometamidium chloride, 50 ng/ml homidium bromide or chloride and 5000 ng/ml diminazene aceturate to kill the entire population in vitro. In contrast, two derivatives of IL 1180 in which resistance to isometamidium had been induced in mice, IL 3343 and IL 3344, required isometamidium chloride at a concentration of 1000 ng/ml and 2000 ng/ml, respectively, to eliminate the populations. The in vitro results showed that the increase in level of resistance to isometamidium in these populations was associated with at least a 200-fold increase in resistance in both populations to homidium, but no increase in resistance to diminazene. KE 2887 and CP 81, two isolates expressing high levels of resistance to both isometamidium and homidium in mice and cattle, were both resistant in vitro to isometamidium chloride and homidium salts at 100 ng/ml. Furthermore, while the former population was resistant to 10,000 ng/ml diminazene aceturate, the latter was sensitive to 5000 ng/ml. IL 3274 and IL 3330, characterised as expressing high levels of resistance to all three drugs in vivo, were shown to be resistant to isometamidium chloride and homidium salts at 1000 ng/ml, and to diminazene aceturate at 10,000 ng/ml. Finally, the in vitro IC100 (concentration of drug required to eliminate 100% of the population) results were consistent with the maximum amounts of each drug detected in vivo.

Animals↗
PubMed1990

Cell number and incidence of chromosomal anomalies in bovine blastocysts fertilized in vitro followed by culture in vitro or in vivo in rabbit oviducts.

The total number of cells and the incidence of chromosomal anomalies in bovine blastocysts cultured in vitro or in vivo in rabbit oviducts were investigated from the four-cell stage after in-vitro fertilization of in-vitro matured follicular oocytes. The total number of cells (80 vs 179) in the oviduct-cultured blastocysts was nearly double that (43 vs 80) of blastocysts cultured in vitro at early and expanded blastocyst stages. In both culture systems, the total number of cells increased with the stage of development. Mitotic index (number of metaphase plates/total number of cells) of blastocysts decreased with development from early (11.5 vs 13.8%) to hatched blastocyst stages (4.8 vs 2.8%) in in-vitro and in-vivo culture systems, respectively. Overall, chromosomal anomalies were observed in 37.5% (27 27 ) of embryos cultured in vitro and in 28.0% (7 24 ) cultured in vivo, respectively. Incidence of chromosomal anomalies did not depend on such factors as culture system or stage of development. Most chromosomal anomalies were polyploid and mixoploid cells.

Journal Article↗
PubMed1991

The interaction of wheat germ tyrosyl-tRNA synthetase and the tRNA-like end of brome mosaic virus RNA has no effect on in vitro viral protein synthesis and on in vitro encapsidation.

The effect of aminoacylation of the tRNA-like end of brome mosaic virus RNA during in vitro protein synthesis and in vitro viral encapsidation was investigated. The components of the homologous system were: BMV RNA, wheat germ cell-free protein synthesizing system and pure tyrosyl-tRNA synthetase from wheat germ. During in vitro protein synthesis directed with tyrosylated as well as non-tyrosylated BMV RNA, no differences were observed in the amount and in the class of polypeptides formed neither in the velocity of the translation reaction. Excess active TyrRS was added during in vitro translation, without modifying the translation efficiency. BMV RNA and active TyrRS were preincubated prior to translation in order to interact without the translation system components and then subjected to translation in vitro. Similar results were obtained when BMV RNA was preincubated with inactive TyrRS or BSA. These results indicate that the aminoacylation of BMV RNA has no pronounced effect on viral protein synthesis in vitro. During BMV RNA encapsidation either tyrosylated or non-tyrosylated BMV RNA 4 could be encapsidated in a similar way.

Capsid↗
PubMed1989

Inhibition of in vitro and in vivo mast cell degranulation by Taenia crassiceps metacestodes in vitro incubation products.

In vitro released products of T. crassiceps metacestodes (TcIP) harvested from the peritoneal cavity of NMRI mice were tested for inhibitory effects on the in vitro degranulation of peritoneal mast cells (MCs) of normal mice (NMRI) and rats (Wistar) and on the in vivo degranulation of rat (Wistar) skin MCs (PCA-assay). In vitro degranulation was elicited chemically (compound 48/80, polymyxin B or the bee venom peptide, mellitin). In vivo degranulation was triggered immunologically (anaphylactic systems ovalbumin/anti-ovalbumin or Fasciola hepatica crude fluke extract antigen/serum of fluke-infected rats (Wistar]. In vitro degranulation of murine peritoneal MCs or the in vitro histamine release of rat peritoneal MCs normally induced chemically was significantly inhibited when the MCs were preincubated with the TcIP or with serum of T. crassiceps-infected NMRI mice from day 35 post infection and thereafter. In vitro degranulation of peritoneal MCs of infected mice was strongly inhibited beginning on day 10 after infection. Also in vivo degranulation of the IgE-sensitized rat skin MCs was significantly reduced by intradermal injection of the TcIP before (6, 3 and 1 h) antigen challenge and by preinjection (1 h) of serum from infected mice (day 80 p.i.)-The inhibitory effect was also demonstrated after immunoadsorption of mouse serum proteins naturally contaminating the TcIP. Heating (100 degrees C/15 min), even in the presence of 0.25 M HCl, did not suppress the inhibitory activity.

Animals↗
PubMed1974

Lymphocyte proliferation in vitro induced by hapten autologous protein conjugates. I. A study on the class of lymphocytes responding in vitro and on the nature and specificity of their receptors.

Immune lymph node cells from guinea pigs respond to soluble antigen in vitro by an increase in DNA synthesis. Optimal conditions for this proliferative response were studied in the present article. Under such conditions, immune cells showed increasing responses with increasing antigen concentration in vitro, the threshold dose of activation frequently being as low as 0.02 microg per culture. In contrast, normal lymph node cells (from FCA-stimulated animals) did only respond to antigen at very high doses (20 mg/culture), and immune cell dilution studies could be performed in normal cells without changing the kinetics of the antigen specific response of immune cells. Fractionation on anti-Ig columns indicated that purified, immune T lymphocytes were quite capable of proliferating in vitro upon antigen stimulation. However, our attempts to adsorb the proliferating cells onto chemically defined immunoadsorbants failed despite the fact that immune B cells (as measured by the rosette assay) were retained almost completely by such a procedure. Purified, immune T lymphocytes from guinea pigs immunized with different antigen concentrations in vivo and/or obtained at different times after immunization were tested for a differential sensitivity toward antigen-induced DNA synthesis in vitro. However, we were not able to demonstrate any regular increase in sensitivity to antigen in vitro, and if found, it seemed to be more dependent upon the number of antigen reactive cells in the population studied rather than upon differences in the average avidity of the receptors on the cells proliferating in vitro. The results in the present article are discussed in relation to current knowledge and hypotheses on T-lymphocyte receptors.

Animals↗
PubMed1975

Genetic control of the in vitro responses of rat blood lymphocytes. I. Comparison of in vitro and in vivo responses.

Nielsen, H. E. & Koch, C. Genetic Control of the In Vitro Responses of Rat Blood Lymphocytes. I. Comparison of In Vitro and In Vivo Responses, Scand. J. Immunol. 4, 31-36, 1975. In vitro DNA synthetic responses of blood lymphocytes from the inbred rat strains BN, Lewis, and AS were compared with each other after stimulation with allogeneic lymphocytes, human lymphocytes, phytohemagglutinin, and anti-Ig antibodies. BN lymphocytes responded less well than AS and Lewis lymphocytes regardless of the stimulus used. The responses of the congenic strains Lewis and Lewis. BN were identical, suggesting that H-1 (Ag-B)-linked loci do not determine in vitro responsiveness in these strains. In vivo, AS and BN rats were equally able to reject third-party skin grafts and to induce graft-versus-host reactions in F1 hybrids. We conclude that the interstrain differences found in vitro reflect genetically determined differences in in vitro kinetics, possibly related to the fraction of short-lived lymphocytes present rather than to demonstrable differences in the frequency of reactive lymphocytes.

Animals↗
PubMed1998

Skin2--an in vitro human skin model: the correlation between in vivo and in vitro testing of surfactants.

The availability of an in vitro test system to replace animal testing of potential irritants is becoming more and more urgent especially in Europe as a consequence of the European Community Cosmetics Directive. To evaluate the ability of Advanced Tissue Sciences' (ATS) ZK1301 skin model to predict the skin irritation potential of surfactants, we performed a pilot validation study utilizing four different laboratories. The in vitro protocol was designed as a quantitative pre-screen for the clinical patch studies. Sixteen substances, representing various surfactant categories and ranges of irritation potential, were tested. The 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to quantitate viability in vitro. We documented the viability of tissues exposed to unknown substances for specific periods. The in vitro results were calculated as percent distilled water controls (DWC). The time required to reduce the viability of each tissue to 50% of the distilled water controls (T50) was compared to mean erythema and edema scores from the clinical studies by Pearson's correlation. The individual laboratories demonstrated coefficients of 0.72. The results indicated that the 30 min percent untreated control values best predicted the 24 h clinical patch scores. No statistically significant interlab variability was found. Only one false negative was seen when non/mild and moderate/severe irritant categories were assigned according to the in vitro scores. These results demonstrate that the skin2 in vitro test system may serve as a good screening method prior to clinical patch studies.

Adolescent↗
PubMed1991

In vitro hypothalamic neurogenesis: morphological maturation of mouse hypothalamic cultures and in vitro versus in situ biochemical analysis.

The morphological and biochemical changes that occur during in vitro neurogenesis of the mouse hypothalamus were studied in rotary cultures prepared from mice between 4 and 16 days of postnatal age. After 6 days of in vitro growth, histotypic cultures with a high degree of morphological differentiation were obtained in cultures prepared from 8- to 10-day-old mice. Before day 8, the cultures showed immature neurons, while after day 12 most of them exhibited an undesired number of degenerated cells. Light-microscopic, Golgi and ultrastructural studies clearly showed the stages of development of the neurosecretory cells in culture. The particular organization of the hypothalamic cells in these cultures can be homologized to its equivalent region in vivo, as demonstrated by their morphological similarities as well as by the fact that the majority of the neurons orient their axons toward the external part of the culture in order to release the neurosecretory material outside the in vitro grown neuronal population, as is the case in situ since hypothalamic neurons release their neurosecretory products at the vascular system. Biochemical parameters such as DNA, RNA and protein contents were determined during the period of in situ development used for the preparation of histotypic cultures and compared to the biochemical changes that occurred during in vitro maturation. The changes in the in vitro DNA and protein contents showed the same variation pattern as in situ. The DNA/protein and RNA/protein ratios also had comparable characteristics, having peak values at days 10 and 16 in situ and in the histotypic cultures prepared from 10-day-old mice. These studies have demonstrated the correlation between the in vitro biochemical and morphological development and the significance of the critical period during hypothalamic neurogenesis for successful organotypic preparations.

Animals↗
PubMed2006

Predictive power of an in vitro system to assess drug interactions of an antimuscarinic medication: a comparison of in vitro and in vivo drug-drug interaction studies of trospium chloride with digoxin.

The authors studied a potential drug-drug interaction via findings from in vitro and in vivo studies, to assess whether the in vitro system was predictive of in vivo clinical pharmacokinetic outcomes. An in vitro experiment and a clinical study were performed to assess the potential for interaction. The effect of trospium chloride on human P-glycoprotein-mediated transport of [3H]-digoxin was determined in vitro. A randomized, crossover clinical trial in 40 subjects was performed to evaluate the effect of trospium on the pharmacokinetics of digoxin in vivo. The findings from the studies were then compared. The in vitro findings in this study were corroborated by the clinical study via assessment of inhibition and impact on pharmacokinetic parameters. The in vitro system for assessment of a potential interaction of 2 drugs excreted primarily through the kidney was predictive of the pharmacokinetic outcomes obtained from a clinical setting.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

In vitro culture conditions using chemically defined media for in vitro matured and intracytoplasmically inseminated porcine oocytes.

The present study investigated in vitro culture methods [droplet and Well of the Well (WOW)] using semi-defined and defined media [modified porcine zygote medium (mPZM)] and the additional effects of insulin on in vitro matured and intracytoplasmically inseminated porcine oocytes. In Experiment 1, in vitro matured and intracytoplasmically inseminated porcine oocytes were cultured for 6 days in the following four groups: 1) mPZM-3 (containing bovine serum albumin) + droplet (30 mul), 2) mPZM-3 + WOW, 3) mPZM-4 (containing polyvinyl alcohol) + droplet, and 4) mPZM-4+ WOW. The culture media (mPZM-3 and mPZM-4) and methods (droplet and WOW) did not significantly affect the cleavage rate, but the blastocyst rate of the oocytes cultured in mPZM-3 was significantly (P<0.01) higher than that of mPZM-4 (20.1 and 9.4%, respectively). The blastocyst rates as percentages of the cleaved oocytes (51.8 and 16.9%) and the hatched blastocyst rate as percentages of the number of blastocysts (12.3 and 2.2%) were also significantly (P<0.01) higher in mPZM-3 compared with those in mPZM-4. There was significant interaction (P<0.05) between the two main factors; the effects of the culture media and methods on the rate of hatched blasyocysts as percentages of the blastocysts produced and, the hatched blastocyst rate (20.3%) as percentages of the number of blastocysts produced in mPZM-3 were significantly (P<0.05) higher than in the other groups. In Experiment 2, the additional effects of insulin (100 ng/ml) in mPZM-3 and mPZM-4 media was investigated in the WOW culture system. Insulin addition did not improve cleavage, blastocyst formation, or the number of cells in blastocysts. However, as in Experiment 1, mPZM-3 resulted in a significantly higher blastocyst rate as percentages of the cleaved oocytes than mPZM-4 (33.9 and 18.4%). These results indicate that a chemically defined medium (mPZM-4) needs to be improved to provide more suitable culture conditions for in vitro development of in vitro matured and intracytoplasmically inseminated porcine oocytes. However, the WOW system may be a useful IVC method for blastocyst development of in vitro matured porcine oocytes following ICSI when a semi-defined medium (mPZM-3) is used.

Animals↗
PubMed1982

The organization, structure, and in vitro transcription of Alu family RNA polymerase III transcription units in the human alpha-like globin gene cluster: precipitation of in vitro transcripts by lupus anti-La antibodies.

We have studied the location, structure, and in vitro transcription of repetitive DNA sequences within the human alpha-like globin gene cluster. At least eight different Alu family repeats were identified, each of which is transcribed in vitro to produce discrete RNA transcripts. The nucleotide sequence of one Alu repeat sequence, located on the 3' side of the alpha l globin gene (3'-alpha l), was determined and compared to published Alu repeat sequences. In vitro transcription of this repeat sequence generates RNA fragments of approximately 410, 260, 160, and 86 nucleotides. To determine whether these transcripts associate with specific proteins in vitro, we carried out immunoprecipitation experiments using an antiserum from systemic lupus erythematosus (SLE) patients. We find that the antiserum anti-La, which was shown to precipitate ribonucleoproteins (RNPs) containing the adenovirus VAI RNA from virus infected cells, preferentially precipitates the smallest two in vitro transcripts of the 3'-alpha l Alu repeat. These results suggest that the RNAs interact with specific factors in the in vitro transcription reaction mix to form RNP.

Antibodies↗
PubMed1985

In vitro maturation of bovine cumulus enclosed primary oocytes and their subsequent in vitro fertilization and cleavage.

Cumulus enclosed primary oocytes from 2 to 4-mm bovine follicles were matured in vitro in Minimum Essential Medium containing follicle-stimulating hormone (0, .1, 1, 10, 50, or 100 micrograms/ml) or human chorionic gonadotropin (0, .1, 1, or 10 IU/ml) for 48 h at 37 degrees C under paraffin oil. Cumulus mass expansion comparable to that seen in vivo occurred in 18% of the control oocytes, 39% of those cultured in human chorionic gonadotropin, and 56% of those cultured in follicle-stimulating hormone. The optimum follicle-stimulating hormone concentration for cumulus expansion was 1 microgram/ml, and this was then used to mature oocytes individually or in groups of 5 for in vitro fertilization. Ejaculated bovine semen, extended 1:10 with yolk-TES-Tris extender and stored 24 to 48 h at 4 degrees C, was warmed, washed once with Minimum Essential Medium, and 500,000 motile sperm/ml were used to inseminate the matured oocyte-cumulus cell complexes. Criteria for fertilization was cleavage to the two-cell stage 48 h after insemination. Oocytes, inseminated individually, cleaved with a frequency of 5%, whereas 15% of those inseminated in groups of 5 cleaved, perhaps as the result of cumulus factors enhancing capacitation. The cleavage rate for the parthenogenetic control with killed spermatozoa was 0%. Therefore, primary oocytes matured in vitro to secondary oocytes were successfully fertilized in vitro and cleaved to at least the two-cell stage in the Minimum Essential Medium. Individual differences between bulls in ability to fertilize in vitro were noted.

Animals↗
PubMed1984

In vitro activation of cyclophosphamide for an in vitro chemosensitivity assay.

The problem of activation of cyclophosphamide (CY) for an in vitro chemosensitivity assay was studied using the B16 melanoma. The efficacy of the S9 hepatic microsomal fraction in vitro was compared with activation by passage of drug in vivo. The effect of CY was assayed by inhibition of tritiated thymidine (3HdThd) uptake by B16 tumor cells in vitro, and by its effect in single dose in vivo on the life span of syngeneic C57BL/6 mice injected with B16 tumor cells. In vivo activation of CY that was achieved by injecting 20 mg of CY intraperitoneally (i.p.) into mice and obtaining plasma 20 minutes later was more rapid and more reproducible, while in vitro activation provided a more potent preparation and a better quantitative correlation with the in vivo effect of CY. The activation activity of different preparations of S9 microsomal fractions was found to be directly related to the activity of aminopyrine demethylase, a mixed function oxidase enzyme, in the S9 fraction, rather than to total protein concentration. The S9-activated drug required 2 1/2 to 3 hours to achieve maximum inhibition of subsequent tumor cell tritiated thymidine (3HdThd) incorporation compared with 20 minutes to achieve maximum inhibition by in vivo activated drug. We conclude that rapid qualitative screening for effectiveness of CY may best be done with in vivo activated drug, whereas quantitative prediction of effective concentration appears to be best achieved with drug activated in vitro by the hepatic S9 fraction.

Aminopyrine N-Demethylase↗