* Drug Safety, Schering-Plough Research Institute, P.O. Box 32, Lafayette, New Jersey 07848-0032;
Preclinical Development and Therapeutic Antibody Discovery, Millennium Pharmaceuticals, Cambridge, Massachusetts 02139;
Drug Metabolism, Schering-Plough Corporation, Kenilworth, New Jersey; and
Drug Safety and Metabolism, Schering-Plough Research Institute, Lafayette, New Jersey 07848
Received August 14, 2001; accepted November 6, 2001
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ABSTRACT |
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Key Words: p53 gene; adenoviral vector; gene therapy; safety evaluation; coagulopathy.
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INTRODUCTION |
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SCH 58500 is administered during clinical trials by several routes, depending on the tumor type. The dose routes include intratumoral administration to breast, melanoma, head and neck, and non-small-cell lung tumors; intraperitoneal (ip) administration to tumors associated with the ovary; and intrahepatic artery (intra-arterial, ia) administration to liver tumors. The eventual therapeutic dosing regimen is anticipated as being a single dose or a small number of daily doses over a short timespan. The dose routes tested in the rodent toxicology studies were intraperitoneal (ip) or subcutaneous (sc). Single-dose and repeat-dose studies were conducted. Testing by the sc route in rats was done to determine the potential for local irritation at the injection site. Intravenous (iv) dosing was done to simulate a worst-case scenario in which the dose was systemically administered.
The adenovirus vector is human (human Ad-5) and potentially immunogenic in laboratory animals. The toxicology program included testing in a nonimmune species, the rat, and an immune species, the Yorkshire pig. Anti-SCH 58500 antibodies, serum neutralizing factors, and gene expression were assayed in several of the rodent studies. Toxicokinetic measurements were either included in the toxicology studies or were conducted separately at the same dose levels as used in the toxicology studies.
Adenoviruses are known to induce hemagglutination of mammalian erythrocytes from some species in in vitro studies. SCH 58500 induces partial hemagglutination of erythrocytes from Sprague-Dawley rats. The toxicological significance of in vitro hemagglutination is not known; consequently a study was conducted to relate hemagglutination in vitro with in vivo toxicity in 2 strains of Sprague-Dawley-derived rats.
Because of the variety of dosing regimens tested, and to avoid repeated descriptions of study procedures, arbitrary study identification numbers were assigned to most studies and are used for reference in the text. The study numbers are listed in Table 1. These studies, in conjunction with pig studies, have supported clinical trials by multiple dosing routes.
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MATERIALS AND METHODS |
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When performed, hematology measurements consisted of erythrocyte, hemoglobin, hematocrit, reticulocyte, platelet, leukocyte, and differential counts. Coagulation measurements included prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and fibrinogen degradation products. Serum chemistry measurements usually included total protein, albumin, globulin, albumin:globulin ratio (A/G), cholesterol, triglycerides, glucose, total bilirubin, urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), alkaline phosphatase, sodium (Na), potassium (K), chloride (CL), calcium, and phosphorous.
Single-dose toxicity studies.
The purpose of the single-dose studies was to identify the general toxic effects of SCH 58500 when administered iv (Study 1 in mice; Study 4 in rats) and ip (Study 2 in mice; Studies 6 and 7 in rats) dose routes. These studies included (when indicated in Table 1) observations for clinical signs of toxicity; body weight, and food consumption; measurements for hematology, coagulation, clinical chemistry, and urinalysis parameters; necropsy observations; organ weights; and microscopic evaluation of tissues. Laboratory measurements were conducted in most studies on the last day of the observation period prior to necropsy and 7 days following dosing in studies with 14-day postdose observation periods. Microscopic examinations of tissues were usually done on tissues from the high-dose and control animals with target organs identified in the high-dose animals being examined at lower dose levels. Some studies included an ophthalmology examination (Studies 3, 6, and 7).
A dose-ranging study by the iv route that was limited in scope was conducted in rats (Study 3) prior to initiation of the single-dose, iv toxicity studies. In mouse Study 2, two batches of SCH 58500 were tested.
To obtain a better understanding of the potential mechanism of toxicity observed in prior studies, we euthanized Study 5 iv-dosed rats at 2, 6, 12, and 24 h after dosing. Hematological and blood chemistry measurements were performed prior to euthanasia.
A study attempting to correlate in vitro hemagglutination observations with in vivo toxicity (Study 8) included Sprague-Dawley-derived rats from 2 sources: Taconic Farms [Tac:N(SD)fBR MPFTM] and Charles River [Crl:CD®(SD)BR VAF/PlusTM]. The rats from Taconic Farms were dosed iv, ip, and sc, and the Charles River rats were dosed by the iv route. Hemagglutination (see Morrissey et al., 2002 for methods) was determined on blood taken from both strains.
To simulate intratumoral injections, a study was conducted primarily to determine local tolerance to SCH 58500 by the sc route. Groups of 6 rats/sex were dosed once (5 ml/kg dose volume) with 0.011, 0.56, or 11 x 1011 pfu/kg, followed by a 14-day observation period. Alternatively, other groups were dosed once daily (1 ml/kg dose volume) for 5 days with the same total dose levels, followed by a 10-day observation period. In addition to local tolerance, hematology and serum chemistry measurements were performed.
Single-dose pharmacokinetic/toxicokinetic and tissue distribution studies.
The disposition of SCH 58500 following iv dosing of mice and rats was usually evaluated in independent studies. Groups of 4 male mice were given iv bolus doses of 0.01, 0.1, 1.1, or 11 x 1011 particles/kg and euthanized for serum and liver samples at 10 and 60 min and 4 days after dosing. Groups of 3 rats were dosed by iv bolus with 3.8 x 1011 pfu/kg and euthanized at 13 time points over 12 h for blood collection to determine pharmacokinetic parameters, and on 7 intervals over 35 days to collect samples of lung, liver, spleen, kidney, and adrenal gland for tissue-distribution evaluations.
In 3 studies separate from the toxicology studies, rats were dosed iv, ip, or sc at the same dose levels used in the single- and repeat-dose studies, (0.011, 0.56, and 11 x 1011 particles/kg) for toxicokinetic and tissue expression evaluations. In the iv and ip studies, blood was collected from 3 rats/sex/group at 0.08, 0.167, 0.25, 0.5, 1, 2, and 4 h postdose. Tissue samples of liver, lung, spleen, kidney, adrenal gland, brain, colon, heart, testes, and mesentery/omentum were collected from 3 rats/sex/group on days 1, 7, 14, 21, and 34 after dosing.
In these studies, tissue polymerase chain reaction (PCR) and plaque formation assays were performed on serum samples to determine SCH 58500-encoded p53 DNA and infectious activity, respectively. Both assays can be used to estimate concentrations of SCH 58500 in serum. Tissues were assayed for SCH 58500-encoded p53 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Plaque-forming assays were performed on rat urine in one iv study to determine if infectious virus was being shed in the urine. The PT-PCR assay was also conducted in (single-dose toxicity) Study 6 on liver, spleen, and mesenteric/omental tissues. The assays were validated with defined levels of accuracy, precision, and sensitivity in several test species.
Repeat-dose toxicity study.
In the repeat-dose study, 10 rats/sex/group were euthanized after 14 or 28 days of ip dosing (Study 10). Observations and measurements included clinical signs, hematology, clinical chemistry, and urinalysis measurements; necropsy observations with organ weights; and a microscopic evaluation of tissues. Most measurements were conducted on days 8, 15, and 29. Urinalysis was conducted on day 8 only. Ophthalmologic examinations were conducted prior to dosing and before sacrifice. Body weight and food consumption was determined weekly and the rats were observed daily for reactions to dosing.
Anti-SCH 58500 antibodies and serum neutralizing antibodies were determined by serum enzyme-linked immunosorbent assay (ELISA) and serum neutralizing factor (SNF) bioassay (an SaOS-2, human osteogenic sarcoma cell line, antiproliferation assay), respectively. RT-PCR assays were conducted from one pooled mesenteric, omental, and liver tissue/sex and 2 pooled spleen tissue/sex/dose group. These samples were collected on the day after the 14th- and 28th-day doses.
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RESULTS |
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In the ip mouse study (Study 2), clinical signs of toxicity differed somewhat between the 2 batches that were tested. Ruffled coat, squinted eyes, and lethargy were observed in mice dosed with 300 x 1011 particles/kg, regardless of batch. At
900 x 1011 particles/kg, ataxia and circling were observed in response to one batch. Cyanosis and deaths (1 mouse/dose level) were observed after
600 x 1011and 900 x 1011 particles/kg of the other batch. All of these observations occurred on the day of dosing. Dose-related, mild (
4%) to moderate (
12%) body-weight losses were observed on the day after dosing of either batch. Dosing with a high volume (3 ml) of vehicle was well tolerated, causing only minimal (
2%) losses in body weight on the day after dosing. In this study, a dose of 3 x 1011 caused no effects and the response to 300 x 1011 particles/kg was generally mild.
Single-dose toxicity in rats.
Signs of toxicity following iv (Studies 3 and 4) and ip (Studies 6 and 7) dosing occurred at the 11 x 1011 particles/kg dose level (Table 2). The highest no-effect dose given iv or ip was considered to be 0.56 x 1011 particles/kg. Antemortem and histopathological observations are tabulated in Tables 2 and 3
, respectively.
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In Study 4, where clinical laboratory measurements were made at 7 and 14 days following dosing, the pattern of changes observed on day 7 was very similar to that observed in Study 3. At 14 days after dosing in Study 4, hematology parameters were comparable to those of the controls. Although serum enzyme levels decreased, they remained somewhat elevated at 14 days postdose. Selected 7- and 14-day clinical laboratory values from Study 4 are tabulated in Table 4. The urine collected from some rats prior to death in Study 4 was acidic and contained large amounts of blood, leukocytes, protein, and urobilinogen.
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The histopathological changes observed in the rats that died acutely after iv dosing with 11 x 1011 particles/kg were more severe. They included, at necropsy, small spleens and dark foci in the thymus and pancreas, and serosa of the stomach, kidneys, and lungs. The livers were mottled and dark red. Microscopic lesions included glomerular capillary fibrin thrombi, renal cortical tubular necrosis, and protein casts in the kidney; congestion, hemorrhage, and necrosis within the spleen; multifocal necrosis within the liver; and hemorrhage and extrinsic vascular thrombi in the pancreas.
When SCH 58500 was given ip at a dose of 11 x 1011 particles/kg, no deaths or significant changes in clinical signs, body weight, food consumption, or hematology and serum-chemistry values, were observed (Studies 6 and 7). Spleen weight was increased in rats euthanized at 7 and 14 days postdose. At necropsy, adhesions and/or discoloration of the liver and spleen were observed in Study 6 but not in Study 7. Adhesions were accompanied by minimal to mild inflammation (mononuclear cell infiltrate, mesothelial proliferation, and fibroplasia) of the serosal surface of multiple abdominal organs. Minimal to mild mononuclear cell infiltrate, increased hepatocellular mitosis, single-cell necrosis, and anisokaryosis in hepatic parenchyma were observed in the liver. A decrease in the degree of observations between 7 and 14 days postdose indicated a trend toward recovery. This may explain why comparable changes were not observed in Study 7, where necropsies were limited to the later interval following dosing. In Study 7, a mild increase in degree but not incidence of extramedullary hematopoiesis was observed in the spleen, and males had an increased incidence of (minimal) serositis relative to that observed in the controls.
There were no SCH 58500-related changes in the ophthalmology examinations in any of the single-dose studies.
Rat mechanism of toxicity study.
In this study, groups of rats were euthanized at various intervals during the initial 24-h period following iv dosing with 11 x 1011 particles/kg (Study 5). Three of 60 rats dosed died within 1 h with no cause of death identified. Clinical signs of toxicity generally included lethargy, soft feces, and a ruffled-hair coat. Selected clinical laboratory measurements are tabulated in Table 5.
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Prothrombin and activated partial thromboplastin times were minimally to mildly higher at all time points. Fibrinogen values were minimally lower at 2 and 6 h and mildly higher at 24 h after dosing. Fibrinogen degradation products of the SCH 58500-dosed rats did not differ from those of the controls.
Changes in serum chemistry values included mild decreases in total protein and albumin at all or most time points, with a decrease in albumin:globulin ratios at 12 and 24 h postdose. Increased serum K and decreased Na and Cl levels were observed only 2 h postdose. Glucose concentrations were mildly to moderately higher at 2 h and decreased at 6 h postdose. Mild increases in BUN occurred at 2, 6, and 12 h and in serum-liver enzymes (AST, ALT, and GGT) at all or most time points. More severe increases in BUN and liver enzyme levels along with an increase in creatinine levels were observed in 2/60 rats.
Increased spleen weights and stress-related increases in adrenals and decreases in thymic weights occurred at 12 and/or 24 h postdose. Edema, hemorrhage, thrombosis, and vasculitis were observed in the pancreas. Stomach lesions included mucosal hemorrhage and necrosis and submucosal edema in the glandular portion. Congestion, hemorrhage, lymphoid atrophy, and lymphoid necrosis were seen in the spleen. Some rats had single-cell necrosis and prominent Kupffer cells in the liver and tubular dilatation, tubular necrosis, and/or casts in the kidneys. These changes were generally progressive and were initially observed at 2 h postdose in the pancreas, liver, and kidneys and at 6 h postdose in the stomach. Stress-induced thymic apoptosis was also observed starting at 2 h postdose. One rat sacrificed at 6 h postdose had mild hemorrhagic necrotizing cecitis and submucosal edema in the colon.
Correlation to hemagglutination.
Partial or complete hemagglutination was observed in vitro with blood collected from rats supplied by both Charles River and Taconic Farms. There was no correlation between acute toxicity and hemagglutination. None of the rats from Taconic Farms died, but 3/14 rats from Charles River were found dead on the day following dosing. Red-colored urine was observed in one rat from Taconic Farms and in 4 rats from Charles River. Two of these rats were among the 3 Charles River rats that died. There were no other significant observations.
Single-dose pharmacokinetic/toxicokinetic and tissue-distribution studies.
In mice dosed intravenously, SCH 58500-encoded p53 DNA (0.11 x 1011 particles/kg and above) and plaque formation (all dose levels) were detected in 10- and 60-min samples but not in the Day 4 samples. The plaque formation values were dose-related and decreased with time (data not shown). Degradation of mouse liver RNA prevented measurement of SCH 58500-encoded p53 mRNA sequencing in liver samples.
Rats given a single iv dose of 3.8 x 1011 particles/kg for PK/TK evaluation showed signs of toxicity consistent with those observed in the single-dose toxicity studies. SCH 58500-encoded p53 DNA and infectious virus (SCH 58500) were rapidly cleared from serum. The t1/2 for serum concentrations of SCH 58500 based on infectious virus was about 7 min. SCH 58500-encoded p53 sequences were detected in several assayed tissues the day after dosing. SCH 58500-specific sequences remained for up to 2 weeks in various tissues but were generally not detected by 3 weeks after dosing.
Toxicokinetic and tissue expression parameters in rats dosed at the same levels used in the toxicity studies are tabulated in Table 6. Rats dosed iv with 11 x 1011 particles/kg had signs of toxicity comparable to those previously described for this dose level. Using the plaque assay, the Cmax at this dose level was 3.9 x 104 pfu/ml with a tmax of approximately 5 min. The AUC was 1.3 x 104 pfu/h/ml with an estimated clearance of 115/h/kg. Serum concentrations calculated using the PCR method (measuring SCH 58500 DNA) were higher. Based on the PCR assay, the Cmax was 2.28 x 106 plaque equivalents (peq)/ml with a tmax of 5 min and AUC of 2.7 x 106 peq/h/ml. Toxicokinetic analyses could not be made at doses of 0.011 and 0.56 x 1011 pfu/kg, because only one quantifiable concentration could be detected at these dose levels. Signs of toxicity were not detected at these dose levels. Positive RT-PCR results were obtained in most tissues prior to DNAse treatment. Only the liver, lung, and spleen were positive for SCH 58500-encoded p53 RNA. No SCH 58500-encoded p53 RNA was detected in testicular samples. Plaque-forming units were not detected in urine, indicating that infectious virus was not being shed in urine.
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Serum concentrations of SCH 58500-encoded p53 DNA determined by the PCR and plaque assays were below the limits of quantitation in almost all samples taken from rats dosed sc with 11 x 1011 pfu/kg. SCH 58500-encoded p53 mRNA was present at the injection sites on the day after injection but not 7 and 14 days following injection. No encoded p53 mRNA was detected in other tissues.
Local tolerance to sc dosing.
Thickening at the injection site occurred in rats dosed with 11 x 1011 pfu/kg, given either once or divided over 5 days. Rats dosed with 0.56 x 1011 pfu/kg over 5 days had a similar thickening. Thickening was observed for 1 to 8 days after injection. Microscopically, chronic inflammation of subcutaneous tissue was observed at the injection sites of most rats given the same dose levels. The degree of inflammation was minimal to mild in rats dosed once and minimal to moderate in rats given these doses over 5 days. Body weight, food consumption, hematology, and serum chemistry measurements were not affected by sc dosing with SCH 58500. No detectable SCH 58500-encoded p53 mRNA sequences were observed in liver samples.
Repeat-dose study.
No SCH 58500-induced changes in clinical signs, body weights, food consumption, ophthalmology examinations, or urinalysis were observed in rats dosed ip for 14 or 28 days. Changes in hematology measurements were variable and were initially observed on day 8 at the 11 x 1011 pfu/kg/day dose level. These changes included minimally lower (generally less than 10% difference from control values) red blood cell counts, hemoglobin concentrations, and hematocrit values. Total white blood cell counts were mildly elevated (less than 65% more than control values), with both mature neutrophil and lymphocyte counts increasing. Minimally higher fibrinogen values were observed by days 15 and 29. Changes in serum chemistry values included higher total protein and globulin and lower albumin concentrations, which occurred mostly at the 11 x 1011 pfu/kg/day dose level.
Spleen weights of rats dosed with 0.56 and 11 x 1011 pfu/kg were higher than those of the controls (mildly and moderately, respectively). Microscopically, inflammation of the peritoneum at the dose sites that extended to the splenic (focal) and pancreatic (diffuse) capsules were observed at the 11 x 1011 pfu/kg dose level. No other microscopic lesions were observed. The no-effect dose level for systemic toxicity was considered to be 0.56 x 1011 pfu/kg/day.
Toxicokinetics of repeat-dose study.
Antibodies capable of recognizing (ELISA) and neutralizing (SNF bioassay) SCH 58500 were observed in a dose-related manner at all dose levels. The values tended to increase as dosing continued from days 14 to 28. Males tended to have higher mean ELISA antibody values than females. The control rats were seropositive for anti-SCH 58500 antibodies by ELISA and SNF bioassay.
SCH 58500-encoded p53 mRNA sequences were detected in one or both pooled spleen tissue samples taken from females at 0.011 x 1011 pfu/kg and above on days 15 or 29. Splenic expression of SCH 58500-encoded mRNA was observed in pooled spleen tissue taken from males dosed with 11 x 1011 pfu/kg (high dose level) on day 15 only. No encoded p53 mRNA sequences were observed in pooled samples of liver or mesenteric/omental tissue.
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DISCUSSION |
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The no-effect dose level following a single iv or ip dose was 0.56 x 1011 particles/kg. Toxicity was dose-related and reasonably consistent from study to study with rats from Charles River. The lowest lethal dose associated with iv dosing was 3.8 x 1011 particles/kg (Study 8), after which approximately 20% of male rats died. At an iv dose of 11 x 1011 particles/kg, 50% of the males and 17% of the females in Study 4 died, but only 10% of males and none of the females in Study 3 died. The differences could not be associated with differences between the batches tested, but as pointed out below, may be related to individual animal differences in sensitivity to SCH 58500. There were no indications that acute toxicity in rats was associated with in vitro observation of hemagglutination or disseminated intravascular coagulation (FDP negative).
The changes in rats observed in Study 4 after a single iv dose of 11 x 1011 particles/kg suggest that deaths at this dose level were most likely caused by renal necrosis resulting from acute glomerular capillary thrombosis. Splenic necrosis and hemorrhage, pancreatic hemorrhage, and multifocal hepatic necrosis were also observed and likely contributed to the death of these rats. Rats that survived this dose level developed acute single-cell hepatic necrosis accompanied by mononuclear cell infiltrates that resolved by day 15, and marked granulomatous inflammation within the spleen.
The sequence of events in the mechanistic study (Study 5) suggested that toxicity was initially manifested as a mild coagulopathy and/or vasculopathy that resulted in consumption of platelets and clotting factors, leakage or loss of intravascular fluid, hemoconcentration, electrolyte and/or fluid shifts, a moderate stress, and/or inflammatory response and a direct or indirect toxic effect on the liver and kidney. Many of the histopathologic observations in this study were also observed in Study 4. However, significant variability in sensitivity to the effects of SCH 58500 was observed among individual rats in Study 5. Red-colored urine and fibrin thrombi within glomerular capillaries observed in Study 4 were not observed in the mechanistic study.
In the repeated-dose ip toxicity study, the absence of clinical signs of toxicity at the high dose (11 x 1011 particles/kg) was consistent with single-dose ip studies and suggests that SCH 58500 causes no direct cumulative effects. The changes in hematology values together with lower albumin and higher fibrinogen concentrations were attributed to a mild inflammatory response. The higher globulin levels correlated with higher SNF and ELISA antibody levels and higher spleen weights, and were considered compatible with a humoral immune response (antibody production) to SCH 58500 that appeared to be greater in males than females. The apparent lower degree of expression of SCH 58500-encoded mRNA in males as compared to females may be related to a greater degree of neutralization by anti-SCH 58500 antibodies in males.
SCH 58500-encoded p53 DNA was present in mice in a dose related manner (starting at 0.11 x 1011 particles/ kg) but the presence of encoded DNA was not sustained between 60 min and 4 days postdose. Plaque-forming units were detected at all dose levels (starting at 0.011 x 1011 particles/kg) for 60 min but not by day 4.
After iv administration, SCH 58500 has a rapid clearance and a potential for high tissue uptake in the rat. Serum concentrations of SCH 58500 based on plaque-forming activity were dose-related and gender-independent. The Tmax of 0.083 h appears to correlate with death observed within one h after dosing in the mechanism study (Study 5). Viral protein (EIA method) was present in urine on the day of dosing only. The absence of infectious material (plaque-forming assay) in the urine of rats suggests that infectious virus was not being shed in urine
After ip administration, absorption of SCH 58500 was delayed with a Tmax of 2 h. Model-dependent estimates of t1/2 ka and T-lag were 0.23/0.31 h and 0.5/0.67 h, respectively, based on the plaque/PCR assays. These values indicate that SCH 58500 is absorbed rapidly after a 30-min delay following ip injection. Serum concentrations of infectious activity (plaque assay) and SCH 58500-encoded p53 DNA were quantifiable for up to 6 h and 4 h after dosing, respectively. SCH 58500-encoded p53 mRNA was present in the liver, lung, spleen, kidney, colon, and mesenteric/omental tissue during the first week of testing and in mesenteric/omental tissue for 2 weeks after dosing.
The absence of p53-encoded DNA in serum and the fact that infectious material was below the limit of quantitation of the plaque assay indicates that the potential for systemic absorption after sc administration is negligible. Although sc administration does not mimic intratumoral injection into a highly vascularized tumor, it does suggest that intratumoral injection of SCH 58500 is likely to result in minimal systemic exposure to SCH 585000.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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MacDonald, J. S., Morrissey, R. E., and Patrick, J. E. (1998). Designing non-clinical safety evaluation programmes for gene therapy products: A personal view. In Safety Evaluation of Biotechnologically-Derived Pharmaceuticals: Facilitating a Scientific Approach (S. A. Griffiths and C. E. Lumley, Eds.), pp. 115128. Centre for Medicine Research International.
Marshall, E. (2000). FDA halts all gene therapy trials at Penn. Science 287,565567.[ISI][Medline]
Morrissey, R. E., Horvath, C., Snyder, E., Patrick, J., Collins, N., Evans, E., and MacDonald, J. S. (2002).Porcine toxicology studies of SCH 58500, an adenoviral vector for the p53 gene.Toxicol. Sci.65,256265.