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PubMed2003

Utility of hepatocytes in predicting drug metabolism: comparison of hepatic intrinsic clearance in rats and humans in vivo and in vitro.

We investigated hepatic in vitro intrinsic clearance (CL(int,in vitro)) in freshly isolated or cryopreserved hepatocytes and compared with CL(int,in vivo) by using nine model compounds, FK1052, FK480, diazepam, diltiazem, troglitazone, quinotolast, FK079, zidovudine, and acetaminophen, in rats and humans. The compounds showed a broad range of in vivo hepatic extraction ratios (rat, 0.05-0.93; humans, 0.03-0.76) and were metabolized by hepatic P450, UDP-glucuronosyltransferase, sulfotransferase, and/or esterase. CL(int,in vitro) was determined from substrate disappearance rate at 1 microM in hepatocytes. CL(int,in vivo) was calculated from in vivo pharmacokinetic data using two frequently used mathematical models (the well stirred and dispersion models). When estimating rat CL(int,in vitro) in freshly isolated hepatocytes, the rat scaling factor values (CL(int,in vivo)/CL(int,in vitro)) showed marked difference among the model compounds (0.2-73.1-fold). The rat CL(int,in vitro) values in freshly isolated hepatocytes were in good agreement with these in cryopreserved hepatocytes. Human CL(int,in vitro) were determined by use of cryopreserved hepatocytes. When human CL(int,in vitro) was regarded as the predicted CL(int,in vivo), the observed and predicted CL(int,in vivo) for FK1052, FK480, troglitazone, and FK079 differed markedly (12.4-199.0-fold). In contrast, using human CL(int,in vitro) corrected with the rat scaling factors yielded better predictions of CL(int,in vivo) that were mostly within 5-fold of the actual values. These results make the evaluation using hepatocytes more useful and provide a basis for predicting hepatic clearance using hepatocytes.

Acetaminophen↗

PubMed2002

Use of energy substrates by various stage preimplantation pig embryos produced in vivo and in vitro.

The aim of in vitro embryo systems is to produce embryos of comparable quality to those derived in vivo. Comparison of embryo metabolism as an indicator of viability may be useful in optimization of culture conditions. The aim of the present study was to determine glucose, glutamine and pyruvate use by various stage pig embryos produced in vitro and in vivo. The results indicate that pig embryos use glucose via glycolysis in significant amounts at all stages examined, regardless of embryo origin. In vitro-derived embryos have significantly increased glycolytic activity after the eight-cell stage, whereas in vivo-derived embryos have increased glycolysis at the blastocyst stage. In vivo-derived embryos have higher rates of glycolysis compared with in vitro-derived embryos. Glucose usage through the Krebs cycle for in vitro- and in vivo-derived embryos increased significantly at the blastocyst stage. Pig embryos produced in vitro used constant amounts of glutamine throughout development, whereas in vivo-derived embryos increased glutamine usage after the eight-cell stage. Pyruvate use was minimal at all stages examined for both in vitro- and in vivo-derived pig embryos, showing significant increases at the blastocyst stage. Krebs cycle metabolism of pyruvate, glutamine and glucose by in vivo-derived embryos was higher than that by in vitro-derived embryos. Current in vitro culture conditions produce pig embryos with altered metabolic activity, which may compromise embryo viability.

Analysis of Variance↗

PubMed1985

Comparison of in vitro and in vivo mitogenic and polyclonal antibody and autoantibody responses to peptidoglycan, LPS, protein A, PWM, PHA and Con A in normal and autoimmune mice.

We have compared the in vitro and in vivo mitogenic and polyclonal antibody (IgM-, IgG-, IgA- and IgM anti-SRBC-secreting PFC) and autoantibody (IgM anti-ssDNA-, anti-bromelin-treated [HB]- and anti-intact mouse RBC-secreting PFC) responses to peptidoglycan (PG), LPS, protein A, PWM, PHA and Con A in young (4-7 weeks) and old (7-8 months) normal (BALB/c, CBA/H, C57BL/6) and autoimmune (NZB, NZB X NZW F1, BXSB, MRL/1; old BXSB and MRL/1 were 4-5 months) mice. Our results demonstrated that: lymphocytes from young and old autoimmune mice (except old BXSB) could be further polyclonally activated in vitro by PG or LPS as well as or better than lymphocytes from young and old normal mice; lymphocytes from young or old autoimmune mice were less polyclonally activated in vitro by protein A or PWM, respectively, than lymphocytes from young or old normal mice; PG and LPS were equally effective polyclonal activators in vitro; in vivo, LPS was a stronger stimulant than PG; in vivo, LPS could induce polyclonal activation in both young and old normal and autoimmune mice, whereas, PG could only induce polyclonal activation in vivo in young and old normal mice, but did not induce further activation in young and old autoimmune mice; only some tests (anti-ssDNA and IgG PFC in vivo, and IgA and anti-HB MRBC PFC in vitro) revealed higher responses in autoimmune than in normal mice, and these higher responses were seen more often in vivo than in vitro; both autoimmune and normal mice had a high frequency of autoantibody (especially anti-ssDNA) secreting cells in polyclonal activation in vitro, whereas a high frequency of these cells in vivo was only found in autoimmune mice; in most cases in vitro, polyclonal activators did not change the frequency of autoantibody and heteroantibody secreting cells, but in vivo, both PG and LPS increased the frequency of anti-ssDNA antibody secreting cells in normal, but not in autoimmune, mice; LPS increased the in vivo, but not in vitro, frequency of cells secreting anti-HB MRBC antibodies in some strains of mice; old mice had lower mitogenic responsiveness than young mice in both autoimmune and normal strains; autoimmune mice had similar, higher or lower mitogenic responses than normal mice, depending on the strain and the age, but in most cases consistent for both B and T cell mitogens; and there was no correlation between the patterns of increased or decreased mitogenic and polyclonal antibody responses in normal and autoimmune mice.4+.

Age Factors↗

PubMed1997

Tissue-specific effects of chlorpyrifos on carboxylesterase and cholinesterase activity in adult rats: an in vitro and in vivo comparison.

Organophosphate (OP) pesticides can bind to carboxylesterase (CaE), which may lower the concentration of OPs at the target site enzyme, acetylcholinesterase (ChE). It is unclear from the literature whether it is the CaE's affinity for the OP and/or the number of CaE molecules which is the dominant factor in determining the protective potential of CaE. We undertook a detailed, in vitro and in vivo survey of both CaE and ChE to ascertain if in vitro sensitivity of CaE and ChE predicted the pattern of inhibition seen after in vivo dosing with chlorpyrifos (CPF; 80 mg/kg, p.o.) in male or female adult Long-Evans rats. For the brain, the in vitro sensitivity to CPF-oxon did predict the in vivo patterns of inhibition: In vitro, brain ChE was approximately 25 times more sensitive to the active metabolite, CPF-oxon, than brain CaE, and in vivo brain ChE was more inhibited than brain CaE. In contrast, the in vitro sensitivity of plasma ChE and CaE did not correlate well with the in vivo pattern of inhibition: In vitro, plasma ChE was approximately 6.5 times less sensitive to CPF-oxon than plasma CaE, but in vivo, plasma ChE was more inhibited than CaE. In order to understand the role of CaE in protecting the brain ChE from inhibition by CPF-oxon in vitro, adult rat striatal tissue was incubated in the presence and absence of adult rat liver tissue and IC50s of CPF-oxon were determined. The increase in the striatal CPF-oxon IC50 value noted for ChE in the presence of liver suggested that CaE was binding the CPF-oxon and limiting its access to ChE. Male liver CaE, which has the same affinity for binding CPF-oxon as female liver CaE but has twice as many binding sites, caused a greater increase in the striatal CPF-oxon IC50 than female liver, suggesting that the number of binding sites does play a role in the detoxification potential of a tissue. In summary, we found that (1) there are tissue and gender-related differences for basal ChE and CaE activity; (2) the in vitro sensitivity of CaE or ChE to CPF-oxon is highly tissue-specific; (3) the pattern of ChE and CaE inhibition after in vivo dosing with CPF is not necessarily predictable from the in vitro IC50 for these same enzymes, and (4) the number of CaE molecules may play a role in modifying the toxicity of CPF.

Animals↗

PubMed1984

Comparison of antigen expression of human renal cancers in vivo and in vitro.

Tissue culture lines have become widely utilized in the study of human cancer. Legitimate questions arise as to the degree of similarity between the in vitro cells and their in vivo counterparts. The author has used eleven monoclonal antibodies (mAbs) of defined specificities to compare antigen (Ag) expression of human renal cancers in vivo and in vitro. Nine of the eleven Ags defined by these mAbs have been previously immunochemically characterized. These mAbs were generated by immunizing mice with human cancer cell lines. Nephrectomy specimens from six patients with adenocarcinoma were available for study as both frozen sections (in vivo) and tissue culture lines (in vitro). Frozen sections were typed by an indirect immunoperoxidase assay while established cell lines were typed by an anti-mouse immunoglobulin rosette assay. Antigens were scored either positive (+) or negative (-). Results revealed three patterns of antigenic expression: (1) consistent expression in vivo and in vitro (gp160, gp115, gp120r, gp120nr, GD3, B2.6, HLA-DR); (2) in vivo (-):in vitro (+) (AJ2, gp40, gp130); and (3) in vivo (+):in vitro (-) (S25). These results suggest that while most antigens (Ags) are consistently expressed or repressed when cells adapt to tissue culture, some Ags, e.g., AJ2, gp40 and gp130, appear to be induced and others, e.g., S25, are suppressed in their new milieu. Moreover, expression of these 11 Ags did not change with up to 40 passages in tissue culture. The demonstrated similarities between in vivo and in vitro cells coupled with the logistic advantages offered by cell lines makes an in vitro system a worthwhile approach to the study of human cancer. However, the fact that differences exist between the two systems underscores the need to extend interesting in vitro findings to the in vivo system prior to making á priori extrapolations or assumptions.

Antibodies, Monoclonal↗

PubMed1995

Cerebellar Purkinje cells from the lurcher mutant and wild-type mouse grown in vitro: a light and electron microscope study.

Lurcher is an autosomal semidominant murine mutation. Lurcher heterozygotes (+/Lc) lose all their cerebellar Purkinje cells by adulthood. Explants from 2 days postnatal (P2) wild-type (+/+) and +/Lc cerebellar cortex were grown in vitro to investigate the role of local neuronal environment and afferent input on the degenerating +/Lc Purkinje cell. In Lurcher explants, Purkinje cells were maintained for up to 25 days in vitro. No significant difference was observed between +/+ and +/Lc Purkinje cell numbers from 10 to 20 days in vitro, as revealed by calbindin-D immunoreactivity. Growing +/Lc explants in association with +/+ explants resulted in no significant difference in Purkinje cell survival (10-20 days in vitro). Image analysis of the gross morphology of calbindin-D-immunostained Purkinje cells from +/+ and +/Lc explants grown in vitro revealed a significant decrease in the total area and dendritic lengths of +/Lc Purkinje cells (15 and 20 days in vitro). The fine structure of +/Lc and +/+ Purkinje cells was examined under the electron microscope (10-25 days in vitro). No difference in ultrastructure was observed between +/Lc and +/+ Purkinje cells grown in vitro, and many features similar to normal Purkinje cell development in vivo were present. These included monosynaptic parallel fibre synapses with Purkinje cell dendritic spines, other interneuron synapses with Purkinje cell dendrites and soma, astroglial investment, and minimal extracellular space in the neuropil. Unusual features observed included a persistence of the perisomatic spines in some Purkinje cells, an absence of Nissl bodies in the Purkinje cell perikaryon, naked Purkinje cell dendritic spines, and occasional heterologous synapses. The results are discussed in the light of previous chimeric analysis of the Lurcher mutation, and a hypothesis is put forward to explain the survival of +/Lc Purkinje cells in vitro.

Animals↗

PubMed1994

Significance of IgG and IgM HCV antibody secretion in vitro in patients with chronic hepatitis C: correlation with disease activity and response to interferon-alpha.

Hepatitis C virus antibodies are found in the serum of most patients with chronic hepatitis C. However, the significance of the humoral response is still uncertain. In this study, in vitro IgG and IgM anti-hepatitis C virus secretion by peripheral blood mononuclear cells of patients with chronic hepatitis C was analyzed. Peripheral-blood mononuclear cells from 21 of 36 patients (58.3%) secreted IgG anti-hepatitis C virus in vitro, as demonstrated with anti-hepatitis C virus-specific enzyme immunoassays and recombinant immunoblot assays. Ten of the 36 patients (27.8%) showed both IgG and IgM anti-hepatitis C virus core in vitro. In 9 of these 10 patients, IgM anti-hepatitis C virus was also detected in serum. Patients with in vitro IgM or IgG anti-hepatitis C virus secretion had higher ALT levels in serum than did patients without such secretion in vitro (99.5 +/- 22.1 and 85.6 +/- 34.4 vs. 38.1 +/- 37.4 U/L; p < 0.0001, p < 0.001). Furthermore, with a histology activity score it was demonstrated that patients with in vitro IgM or IgG HCV antibodies (or both) had more severe chronic active hepatitis than did patients without in vitro hepatitis C virus antibody secretion (p < 0.01). To analyze the therapy outcome, we included in this study 18 patients who had received interferon-alpha previously. Seven of eight in vitro hepatitis C virus antibody-positive patients were nonresponders, whereas the in vitro hepatitis C virus antibody-negative patients were mostly complete therapy responders (8 of 10).(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine Transaminase↗

PubMed2001

A semiparametric deconvolution model to establish in vivo-in vitro correlation applied to OROS oxybutynin.

In vitro-in vivo correlation (IVIVC) models may be used to predict in vivo drug concentration-time profiles given in vitro release characteristics of a drug. This prediction is accomplished by incorporating in vitro release characteristics as an input function (A(vitro)) to a pharmacokinetics model. This simple approach often results in biased predictions of observed in vivo drug concentrations, and it can result in rejecting IVIVC. To solve this problem we propose a population IVIVC model that incorporates the in vitro information and allows one to quantify possibly changed in vivo release characteristic. The model assumes linear kinetics and describes the in vivo release as a sum of A(vitro) and a nonparametric function (A(d), a spline) representing the difference in release due to in vivo conditions. The function A(vitro) and its variability enter the model as a prior distribution. The function A(d) is estimated together with its intersubject variability. The number of parameters associated with A(d) defines the model: no parameters indicates perfect IVIVC, a large number of parameters indicates poor IVIVC. The number of parameters is determined using statistical model selection criteria. We demonstrate the approach to solve the IVIVC problem of an oral extended release oxybutynin form (OROS), administered in three pharmacokinetic studies. These studies present a particular challenging case; that is, the relative bioavailability for the OROS administration is >100% compared with that of the immediate-release form. The result of our modeling shows that the apparent lack of IVIVC can be overcome: in vivo concentration can be predicted (within or across data sets) based on in vitro release rate together with a simple form of systematic deviation from the in vitro release.

Biological Availability↗

PubMed2004

Interpretation and optimization of the dissolution specifications for a modified release product with an in vivo-in vitro correlation (IVIVC).

This article considers the in vivo significance attached to in vitro dissolution testing. Almost invariably, the in vitro dissolution test is interpreted in terms of bioequivalence. The literature that describes methods for setting in vitro dissolution specifications is reviewed. The most common interpretation of these specifications is a deterministic one, that is, those batches passing the dissolution specifications would be bioequivalent with the reference if tested in vivo and those failing the dissolution specifications would not be bioequivalent if tested in vivo. Due to random variation, the deterministic interpretation is not appropriate. Instead, we need to consider the conditional probability that a batch that has passed the in vitro dissolution test would demonstrate bioequivalence if tested in vivo, and that a batch known to have failed the in vitro dissolution test would demonstrate bioinequivalence if tested in vivo. One way to estimate these probabilities is by means of a simulated experiment in which the production and testing (in vivo and in vitro) of a large number of batches is computer simulated. Such a simulation can only be performed if the relationship between the in vitro dissolution characteristics and the in vivo performance of the product has been modeled. These models are generally referred to as in vivo-in vitro correlations (IVIVC). The results of one such experiment are described. The above-mentioned conditional probabilities are shown to depend on the choice of dissolution specifications. This result leads to the notion of optimal dissolution specifications. However, both of the conditional probabilities cannot be maximized simultaneously. The probability of making a correct decision on the basis of the in vitro dissolution test is introduced as a possible optimality criterion. This probability is a linear combination of the two conditional probabilities of interest. Using this criterion, the optimal dissolution specifications can be found by searching over the multidimensional space defined by the half width of each interval used in the specifications to find the combination that maximizes this probability. This process is demonstrated using the Nelder-Mead search routine. The choice of dissolution specifications has profound implications for the routine production of the product because if the specifications were very narrow the probability of a batch passing would be low, resulting in a low hit rate. The same computer program used to perform the simulation experiment can be used to estimate the hit rate. Furthermore, it can be used to explore the magnitude of changes required in the parameters describing the test product (particularly variability) to increase a low hit rate to an acceptable level.

Biological Availability↗

PubMed1995

Oesophagostomum dentatum: population dynamics and synthesis of prostanoids by histotropic stages cultured in vitro.

The dynamics of production and excretion of prostanoids by histotropic larvae of Oesophagostomum dentatum and the role of prostanoids for the development of O. dentatum were studied under in vitro conditions. In control cultures fourth stage larvae (L4) were first seen on Day 7 of in vitro cultivation. Their numbers steadily increased until Day 21 of in vitro cultivation. Thereafter, the numbers of L4 no further increased. The continuous presence of inhibitors of prostanoid synthesis such as acetylsalicyclic acid (ASA, 5.6 mmole/liter) or indomethacin (INDO, 1.4 mmole/liter) in the culture medium blocked the development to L4 almost completely. When ASA was given first on Day 13 of in vitro cultivation L4 counts still increased during the following 4 days. However, the numbers of larvae which reached the L4 stage in these cultures was reduced to 65% of that in control cultures. Resumption of development to L4 was seen after the withdrawal of INDO in cultures treated with this drug until Day 13 of in vitro cultivation. Larvae and supernatants were collected separately on Days 7, 14, 21, or 28 of in vitro cultivation and larvae were homogenized. The presence of prostaglandin (PG) E2, PGD2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (TX) B2 was assayed radioimmunologically in homogenates and supernatants. In the homogenates only PGE2 and PGD2 were regularly detected. The total amount of PGE2 and PGD2 in the homogenates significantly increased with the increase in L4 numbers, whereas the homogenate protein-based values did not. In addition to PGD2 and PGE2, PGF2 alpha and TXB2 were found in the supernatants. PGE2 accounted for about 50% of the prostanoids in the supernatants on Day 7 of in vitro cultivation but was generally absent thereafter. After 14, 21, and 28 days of in vitro cultivation TXB2 was the most prominent single prostanoid in the supernatants. PGD2 was found in the supernatants only after 7 and 28 days of in vitro cultivation. PGF2 alpha was present in decreasing amounts in the supernatants throughout the cultivation period. In conclusion, histotropic larvae of O. dentatum produce and excrete mediators of the prostanoid type which may serve as endogenous hormone-like growth factors.

Animals↗

PubMed2002

Association between in vitro platinum resistance in the EDR assay and clinical outcomes for ovarian cancer patients.

OBJECTIVE: The initial clinical response to platinum is a major determinant of outcome for patients with ovarian cancer. This retrospective study was undertaken to correlate the response and survival of newly diagnosed advanced ovarian cancer patients who received platinum-based therapy with in vitro drug response to cisplatin or carboplatin measured as percentage cell inhibition (PCI) in the in vitro Extreme Drug Resistance (EDR) assay. METHODS: Outcomes for newly diagnosed ovarian cancer patients with tumor specimens submitted in a serial fashion for the EDR assay were studied. EDR assay results for cisplatin and carboplatin were correlated with clinical outcome for 79 evaluable chemotherapy nai;ve cases who presented with advanced (stages IIC, III, and IV) ovarian cancer. Stage IV and suboptimally debulked stage IIIc accounted for 16 cases, while 63 cases were optimally debulked Stage III/IIc. All patients were treated with platinum-based combination chemotherapy at a single institution. In vitro results for patient tumors were classified as low drug resistance (PCI > median), intermediate drug resistance [PCI between the median and 1 standard deviation (SD) below the median], or extreme drug resistance (PCI more than 1 SD below the median). For the purpose of this analysis, in vitro EDR to either cisplatin or carboplatin was considered to represent extreme resistance to platinum (EDRP), while the absence of EDR to either cisplatin or carboplatin was considered to represent low resistance to platinum (LDRP). Patients demonstrating relative in vitro resistance to paclitaxel and non-cross-resistance to cyclophosphamide and/or doxorubicin received cyclophosphamide plus platinum (CP); cyclophosphamide, doxorubicin, and platinum (CAP); or platinum alone in place of paclitaxel plus platinum (TP). Progression-free survival (PFS) and overall survival (OS) were correlated with EDR assay results. RESULTS: Median PFS was 6 months for the 17 cases exhibiting EDRP, compared to 24 months for the 62 cases exhibiting LDRP in vitro [relative risk (RR) 3.78, confidence intervals (CI) 1.82-7.83], adjusted for stage, debulking status, in vitro response to 3-OH-cyclophosphamide, and histological grade. Estimated overall 5-year survival was 19% for patients with tumors showing EDRP, compared to 68% for patients with tumors showing LDRP (RR 2.32, CI 1.06-5.07). Patients treated with CP (n = 20) showed no significant difference in OS compared to patients treated with TP (n = 54), CAP (n = 4), or cisplatin (n = 1) alone. In vitro platinum response remained an independent predictor of PFS and OS in multivariate analyses adjusted for CP versus TP, CAP, or platinum administration, and adjusted for debulking status. Median PFS for all 79 patients was 22 months, with an estimated 5-year survival of 57%. CONCLUSIONS: Patients with tumors demonstrating in vitro EDR to platinum were at significantly increased risk for progression and death when treated with standard platinum-based regimens. Such patients may therefore benefit from entry onto trials with novel agents or combinations.

Antineoplastic Agents↗

PubMed1999

In vitro antibody-dependent enhancement assays are insensitive indicators of in vivo vaccine enhancement of equine infectious anemia virus.

We have previously demonstrated a high propensity for enhancement of virus replication and disease resulting from experimental immunization of ponies with a baculovirus recombinant envelope (rgp90) vaccine from equine infectious anemia virus (EIAV). The current studies were undertaken to examine the correlation between the observed in vivo vaccine enhancement and in vitro assays for antibody-dependent enhancement (ADE) of EIAV replication. Toward this goal an optimized EIAV in vitro enhancement assay was developed using primary equine macrophage cells and used to evaluate the enhancement properties of immune serum taken from rgp90 immunized ponies that displayed various levels of vaccine enhancement after experimental challenge with EIAV. For comparison, we analyzed in parallel immune serum samples from a group of ponies immunized with a viral envelope subunit vaccine (LL-gp) that produced sterile protection from EIAV challenge. The results of these assays demonstrated that the rgp90 immune serum had a greater propensity for in vitro enhancement of EIAV replication than serum from the protected LL-gp immunized ponies; in vitro enhancement levels for the rgp90 immune sera averaged about 1.5, with a maximum enhancement value of about 2.0. While distinguishing between immune serum produced by the rgp90 and LL-gp immunizations, the in vitro enhancement assay failed to reliably correlate with the severity of in vivo enhancement observed among the rgp90 vaccine recipients. Vaccinated ponies that experienced moderate to no disease signs displayed levels of in vitro enhancement similar to those of ponies that experienced severe and fatal enhancement of disease after viral challenge. The observed in vitro enhancement was demonstrated to be dependent on serum immunoglobulin, but independent of complement. These studies demonstrate in the EIAV system that in vitro ADE assays appear to be relatively insensitive indicators of the severity of in vivo enhancement and that relatively low levels of in vitro ADE can be associated with severe to fatal enhancement of virus replication and disease in vivo. These observations suggest that relatively low levels of serum ADE observed in other lentivirus systems, including HIV-1, may have more profound effects on in vivo virus replication and disease than previously recognized.

Animals↗

PubMed2001

Novel approach to analysis of in vitro tumor angiogenesis with a variable-pressure scanning electron microscope: suppression by matrix metalloproteinase inhibitor SI-27.

Degradation of basement membrane by metalloproteinases (MMP) is a critical step in tumor angiogenesis. To evaluate in vitro angiogenesis, several models have been employed, including bovine cornea, fenestrated rat brain, Matrigel, and others. These models did not provide quantitative analysis of capillary formation. The current study aimed for a novel approach to in vitro assay of angiogenesis with a "wet scanning electron microscope (SEM)" to investigate suppression of tumor angiogenesis by the MMP inhibitor, SI-27. The effects of noncytotoxic concentrations of SI-27 (1-100 microM) were determined on nonmitogenic vascular endothelial growth factor (VEGF) (10 ng/ml)-mediated cell motility and in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs). Activities of MMP and tissue inhibitor of metalloproteinase (TIMP) were determined by enzyme-linked immunosorbent assay (ELISA). Subsequently, the inhibitory effect of SI-27 was examined on in vitro angiogenesis stimulated by supernatants of human glioma cell lines (U87MG, U251MG, or U373MG). In vitro angiogenesis was quantitatively analyzed with a variable-pressure SEM. Cell motility and in vitro angiogenesis by HUVECs were significantly increased by VEGF along with elevated MMP-1 and -2 activity, whereas SI-27 significantly suppressed VEGF-mediated in vitro angiogenesis and inactivated both MMP-1 and MMP-2, but not inhibited cell motility. The angiogenesis promoted by glioma supernatants showed a significant reduction in the presence of SI-27. SI-27, a novel MMP inhibitor, inhibited tumor angiogenesis in vitro. It can be anticipated to prevent tumor growth through its angiosuppressive effect. Quantitative analysis with a variable-pressure SEM is a novel approach to in vitro angiogenesis assay.

Angiogenesis Inhibitors↗

PubMed2000

Effects of gemcitabine and araC on in vitro DNA synthesis mediated by the human breast cell DNA synthesome.

PURPOSE: Gemcitabine (dFdC) and cytarabine (araC) are both analogs of deoxycytidine. Gemcitabine is a relatively new drug that has been shown in both clinical trials and in vitro systems to have more potent antitumor activity than araC. We have previously isolated a fully functional multiprotein DNA replication complex from human cells and termed it the DNA synthesome. Using the DNA synthesome, we have successfully examined the mechanism of action of several anticancer drugs that directly affect DNA synthesis. In this study, we compared the effects of dFdC and araC on in vitro DNA synthesis mediated by the DNA synthesome with the effects of these drugs on intact MCF7 cell DNA synthesis. METHODS: We examined the effects of dFdC and araC on intact MCF7 cell DNA synthesis and clonogenicity. We also performed in vitro SV40 replication assays mediated by the MCF7 cell-derived DNA synthesome in presence of dFdCTP and araCTP. The types of daughter molecules produced in the assay were analyzed by neutral and alkaline agarose gel electrophoresis. Finally, we examined the effects ofdFdCTP and araCTP on the synthesome-associated DNA polymerase alpha and delta activities. RESULTS: Our results showed that dFdC was more potent than araC at inhibiting intact MCF7 cell DNA synthesis and clonogenicity. [3H]Thymidine incorporation was inhibited by 50% at a dFdC concentration of 10 microM, which was about tenfold lower than the concentration of araC required to inhibit intact cell DNA synthesis by the same amount. As examined by clonogenicity assay, dFdC was also significantly more cytotoxic than araC after a 24-h incubation. In vitro SV40 replication assays using the DNA synthesome derived from MCF7 cells demonstrated that the formation of full-length DNA along with replication intermediates were inhibited by dFdCTP in a concentration-dependent manner. Full-length DNA was produced in the in vitro DNA replication assay even when the dFdCTP was incubated in the assay at concentrations of up to 1 mM. We observed that in the presence of 10 microM dCTP, 3 microM dFdCTP and 60 microM araCTP were required to inhibit in vitro SV40 DNA synthesis by 50%. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there was no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome-associated DNA polymerases alpha and delta. It was found that the drug concentrations required to inhibit 50% of the synthesome-associated DNA polymerase delta activity were much higher than those required to inhibit 50% of DNA polymerase alpha activity for both dFdCTP and araCTP. CONCLUSION: Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase alpha activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis.

Antigens, Polyomavirus Transforming↗

PubMed1994

Developmental ability of porcine oocytes matured and fertilized in vitro.

The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42 71 ) and with monospermic penetration and male and female pronuclei (32%, 23 71 ) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18 71 and 8%, 6 71 , respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28 288 and 13%, 41 318 , respectively) were also higher (P < 0.05) than in controls (2%, 6 284 and 6%, 16 248 , respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9 288 in PFF-treated group and 2%, 6 284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3 100 ) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.

Journal Article↗

PubMed1984

Temperature-mediated processes in teleost immunity: differential effects of in vitro and in vivo temperatures on mitogenic responses of channel catfish lymphocytes.

The in vitro mitogenic responses of channel catfish peripheral blood leucocytes to ConA and LPS were differentially affected by both in vitro and in vivo temperatures. The magnitude of the response to LPS was relatively independent of both in vitro culture temperature and in vivo acclimation temperature. The magnitude of the response to ConA was suppressed at lower in vitro temperatures although this suppression could be reduced by lower in vivo acclimation temperatures. In vitro temperature-shift experiments indicated that channel catfish PBL could respond to ConA at a lower in vitro temperature if first stimulated with ConA at a higher in vitro temperature. The converse, however was not true in that channel catfish PBL did not respond at a higher in vitro temperature after an initial stimulation with ConA at a lower in vitro temperature. This latter failure to respond could not be attributed to the induction of a suppressor cell (or factor) by exposure to ConA at a lower temperature. These studies, when coupled with other available data on channel catfish PBL subpopulations, are interpreted as supporting the hypothesis that low temperature immunosuppression in fish may result from preferential inhibitory effects on T cells rather than B cells.

Animals↗

PubMed2004

In vivo and in vitro evaluation of three controlled release principles of 6-N-cyclohexyl-2'-O-methyladenosine.

6-N-Cyclohexyl-2'-O-methyladenosine was formulated into controlled release formulations exhibiting comparable in vitro release profiles using different formulation principles, i.e. osmotic pump tablets, membrane-coated pellets and hydrophilic matrix tablet. Dissolution behaviour of these formulations was evaluated in vitro under various testing conditions to assess the effect of pH and hydrodynamic conditions. It was found that osmotic tablets were not sensitive to dissolution media pH and hydrodynamics change, while drug release from monolithic hydrophilic matrix tablets were pH-dependent. When tested in vivo in dogs, it was found that metabolism of 6-N-Cyclohexyl-2'-O-methyladenosine was extensive and appeared to be saturable based on a pharmacokinetic study. Cumulative percent input in vivo (%dose) was obtained by numerical deconvolution, and compared to in vitro release profiles. A linear correlation between fraction absorbed (FRA) in vivo and fraction dissolved (FRD) in vitro was established for osmotic tablets--a true zero-order release formula, whereas only a nonlinear correlation was obtained for membrane-coated pellets. The difference in the in vivo behaviour of these formulations, despite their similar in vitro release characteristics, demonstrated the effect of different controlled release principles on their in vivo bioavailability. The curvature of fraction absorbed in vivo vs. fraction dissolved in vitro for membrane-coated pellets indicated that there was a time-scale difference between in vivo and in vitro testing. In conclusion, drug release from the osmotic system was independent of in vitro and in vivo conditions, where best sustained release effect was achieved, whereas the in vitro dissolution test employed for membrane-coated pellets and hydrophilic matrix tablets needed to be optimized to be biorelevant.

Adenosine↗

PubMed1998

Effect of luteinizing hormone (LH), PGE2, 8-EPI-PGE1, 8-EPI-PGE2, trichosanthin, and pregnancy specific protein B (PSPB) on secretion of progesterone in vitro by corpora lutea (CL) from nonpregnant and pregnant cows.

Secretion of progesterone by Day 14 bovine corpora lutea (CL) of the estrous cycle and Day 200 CL of pregnancy was evaluated in vitro to determine what regulates secretion of progesterone by CL of pregnancy. Weights of Day 200 CL of pregnancy (4356 +/- 223 g) were heavier when compared to Day 14 CL of the estrous cycle of Brahman cows (3643 +/- 128 g; p < or = 0.05); however, both Day 14 and Day 200 minced CL slices secreted similar basal amounts of progesterone per unit mass (p > or = 0.05). Secretion of progesterone in vitro by Day 14 CL of the estrous cycle was increased at 4 and 8 h (p < or = 0.05) by 10 or 100 ng/mL luteinizing hormone (LH) and did not differ between doses (p > or = 0.05). Progesterone secretion in vitro by Day 200 CL of pregnancy was not increased (p > or = 0.05) by LH at 4 or 8 h. However, progesterone secretion in vitro by Day 14 CL of the estrous cycle or Day 200 CL of pregnancy was increased (p < or = 0.05) at 4 h by 10 or 100 ng/mL PGE2, which did not differ by dose or reproductive status (p > or = 0.05). At 8 h, Day 14 CL of the estrous cycle secretion of progesterone in vitro was increased (p < or = 0.05) by both doses of PGE2 but only at 8 h by 100 ng/mL from Day 200 CL of pregnancy (p < or = 0.05). Secretion of progesterone in vitro was not affected (p > or = 0.05) by 10 or 100 ng/mL 8-Epi-PGE1 or 8-Epi-PGE2 at 4 or 8 h from Day 14 CL of the estrous cycle or Day 200 of pregnancy. Trichosanthin increased (p < or = 0.05) secretion of progesterone in vitro by 10 ng/mL at 4 h and at 8 h by Day 14 CL of the estrous cycle or at 8 h by Day 200 CL of pregnancy but trichosanthin at 100 ng/mL did not affect (p > or = 0.05) secretion of progesterone in vitro by Day 14 CL of the estrous cycle or Day 200 CL of pregnancy at 4 or 8 h. Pregnancy specific protein B (PSPB) increased (p < or = 0.05) secretion of progesterone in vitro at 4 and 8 h by Day 14 CL of the estrous cycle and did not differ between incubation times (p > or = 0.05). PSPB increased secretion of progesterone at 4 h but not at 8 h (p > or = 0.05) by Day 200 CL of pregnancy. These data suggest that PGE2 or PSPB but not LH, 8-Epi-PGE1 or 8-Epi-PGE2 regulates luteal secretion of progesterone by bovine CL at mid-pregnancy. In addition, it is suggested that weights of bovine CL of pregnancy increase to compensate for a lack of placental secretion of progesterone.

Animals↗