Porcine Toxicology Studies of SCH 58500, an Adenoviral Vector for the p53 Gene

Richard E. Morrissey*,1, Christopher Horvath{dagger}, Eileen A. Snyder*, James Patrick{ddagger}, Nathaniel Collins*, Ellen Evans* and James S. MacDonald§

* Drug Safety, Schering-Plough Research Institute, P.O. Box 32, Lafayette, New Jersey 07848-0032; {dagger} Preclinical Development and Therapeutic Antibody Discovery, Millennium Pharmaceuticals, Cambridge, Massachusetts 02139; {ddagger} Drug Metabolism, Schering-Plough Corporation, Kenilworth, New Jersey 07033; and § Drug Safety and Metabolism, Schering-Plough Research Institute, Lafayette, New Jersey 07848-0032

Received August 13, 2001; accepted November 6, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenoviral vectors are being actively investigated for their potential utility in gene therapy. SCH 58500, a replication-deficient adenoviral vector, carries the normal p53 tumor suppressor gene, which is frequently mutated or absent in several human cancers. To assess the potential toxicity associated with adenoviral use, Yorkshire pigs were dosed by intravenous, intrahepatic, or local routes (subcutaneous and intradermal) to support a variety of potential clinical indications. Porcine cells were shown to support replication of wild-type human adenovirus. The nonlethal and asymptomatic dose in pigs following dosing via the intrahepatic route was greater than 3 x 108 plaque-forming units (pfu)/kg (2.2 x 1011 particles/kg), but less than 2.1 x 109 pfu/kg (1.5 x 1012 particles/kg). By the intravenous route it was 1 x 108 pfu/kg, and by the ip route it was greater than or equal to 3 x 108 pfu/kg. In a multicycle intraperitoneal study in pigs, the high dose of 3 x 108 pfu/kg caused an increased antibody and/or an inflammatory response. By the intravenous route, plaque-forming units were present in most pigs at 5 min postdose, but only in a few at 10 min postdose. No expression was found in gonadal tissue approximately 3 weeks after a single intravenous injection of 3 x 108 pfu/kg. At high intrahepatic doses (about 1.5 x 1012 particles/kg), acute cardiovascular and hemodynamic effects were found, which in subsequent studies were also present at high doses by intravenous administration. Based on these findings, careful evaluation of hemodynamic parameters in patients receiving systemic doses of SCH 58500 is warranted.

Key Words: p53 gene; adenoviral vector; Yorkshire pigs; gene therapy; safety evaluation; toxicokinetic evaluation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SCH 58500 is a recombinant, replication-deficient adenoviral vector containing the cloned human wild-type (normal) tumor suppressor gene p53 (wtAD53). Loss of the wild-type p53 function in cells can lead to uncontrolled growth and development of several tumor types (Donehower et al., 1992Go; el-Deiry et al., 1993Go; Hinds and Weinberg, 1994Go; Hollstein et al., 1991Go; Kuerbitz et al., 1992Go; Lane, 1992Go; Lowe et al., 1994Go; Prives, 1994Go; Vogelstein and Kinzler, 1992Go). The reintroduction and expression of p53 in these tumors can suppress tumor growth and induce apoptosis (Chen et al., 1990Go; Frebourg et al., 1994Go; Shaw et al., 1992Go; Wills et al., 1994Go; Yonish-Rouach et al., 1991Go). The vector for the p53 gene is a human adenovirus (human Ad-5) that was made replication-deficient by deletion of the adenoviral E1 region to minimize the potential for adverse effects associated with its replication.

SCH 58500 is undergoing clinical trials for the treatment of tumor types that commonly express mutant p53 phenotypes. In these trials the drug is delivered by several routes, depending on the tumor type. The dose routes include: intratumoral administration to breast, melanoma, head, neck, and non-small-cell lung tumors; intraperitoneal (ip) administration to tumors associated with the ovary; and hepatic artery (intra-arterial, ia) administration to liver tumors. The therapeutic dosing regimen is anticipated as being a single dose or a small number of daily doses over a short time span, or possibly cyclic in combination with chemotherapy. Consequently, drug safety studies were designed to support the clinical program within the limits of the animal models.

The purpose of this report is to describe the toxicology program that was conducted in support of the safety of SCH 58500. The design of the program considered potential similarities and differences in responses to SCH 58500 between humans and laboratory animals. The adenovirus vector (human Ad-5) is potentially immunogenic; most humans are seropositive to this adenovirus. A herd of Yorkshire pigs was identified with spontaneous development of antibodies that cross-react with SCH 58500. Due to the potential for replication-competent adenoviruses to be in the drug at low levels, studies were conducted to determine the effect of replication-competent adenovirus in pigs and also to demonstrate that Yorkshire pigs with presumed immunity against SCH 58500 were permissive hosts (Betts et al., 1962Go; Torres et al., 1996Go) for human wild-type adenovirus-5 (wtAD-5). Anti-SCH 58500 antibodies and serum neutralizing factors were determined in each study. The potential for gene expression in the test species was determined by tissue expression measurements in most studies. A specific test for expression of p53 in gonadal tissue was conducted to evaluate the possibility of integration in germ-line tissue. Toxicokinetic measurements were included in most studies to measure the rate of clearance of SCH 58500 relative to biological effects. Because observations in an early single-dose general toxicity study suggested a potential cardiovascular effect in response to high doses, cardiovascular/hemodynamic studies were also conducted.

Single-dose and repeated-dose toxicity studies were conducted. The dose routes tested included ip and ia routes that simulated proposed clinical dosing methods directed at tumor targets. Intradermal (id) and subcutaneous (sc) dosing were done to evaluate the potential local toxicity that may occur from intratumoral dosing. Since local or regional dosing of target organs or tissues is expected to be systemically distributed, testing by the iv route was done to simulate a worst-case scenario for unintended systemic circulation resulting from intratumoral injection or inadvertent iv injection. We have previously reported results of intravenous and intrahepatic artery toxicity studies (Morrissey et al., 1997Go).

To minimize potential for confusion and the need for repeated descriptions of study procedures, arbitrary study identification numbers were assigned to all in vivo studies. These numbers, along with protocol summaries, are listed in Table 1Go. The study numbers are used when appropriate as references in the text. In addition to the Yorkshire pig, the rat was used as a second (rodent) test species (Morrissey et al., 2002Go).


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TABLE 1 Protocol Outlines of Most Studies of SCH 58500 Conducted in Yorkshire Pigs
 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
General procedures.
The domestic Yorkshire pigs (Sus scrofa) used in most studies ranged in age from 6 to 16 weeks (adult sows and boars were used in a gonadal expression study). The pigs weighed approximately 20 to 40 kg at the start of dosing and were acclimated for a minimum of 5 days prior to the start of a study or following catheter implantation for dosing (iv, ia, ip). Pigs were generally monitored for clinical pathology endpoints for about a week prior to dosing. They were fed twice daily with Purina Porcine Laboratory Grower 5084TM and had free access to tap water. All pigs used in these studies tested positive for antibodies capable of binding to SCH 58500 and for serum-neutralizing factors, prior to initiation of dosing.

Anti-SCH 58500 antibodies and serum-neutralizing antibodies were determined by serum enzyme-linked immunosorbent assay (ELISA) and serum neutralizing factor (SNF) bioassay (a SaOS-2, human osteogenic sarcoma cell line, antiproliferation assay), respectively. Serum plaque assays (using a cell line that allowed for replication of the replication-defective vector) and serum and tissue polymerase chain reaction (PCR) assays were used for quantitation of virus and SCH 58500-encoded p53 DNA in serum and tissues, respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted to determine the presence of SCH 58500-encoded p53 sequence in portions of selected tissues. For these assays, typical intervals for collection of serum were at 14 time points over 12 h and on 1, 3, and 14 days after dosing for tissues (usually the liver, lung, spleen, kidney, adrenal gland, brain, colon, ovary, and heart). Pigs were surgically catheterized to allow serial blood collection with minimization of stress. All assays were appropriately validated for use with pig samples.

The dose levels of SCH 58500 are primarily identified as particles/kg. For comparison, 7.5 x 1010 particles/kg is approximately equal to 1 x 108 plaque-forming units (pfu)/kg. Control groups were included in all appropriate studies. The controls were dosed with sucrose or sucrose/glycerol/polysorbate-based vehicle except in Study 3, where saline was used as the control substance. A tabulated summary of dose groups and observations and measurements in the single-dose and repeated-dose studies is given in Table 1Go.

Host permissiveness study.
Seven pigs were dosed with human wild-type Ad-5 by infusion of 2.2 x 1011 particles/kg into the hepatic artery (Study 1). In addition to the measurements identified in Table 1Go, sera and tissues for viral culture were taken on the day of dosing (~12 h after dosing) and on 1, 2, and 7 days after dosing. Samples were homogenized and one ml was added to the corresponding flask containing HeLa S3 cells and incubated for 7 days for viral amplification. HeLa S3 cells were then lysed by 5 freeze-thaw cycles and passed to A54a indicator cells. The cultures were incubated for 14 days without refeeding and observed for cytopathic effect, which was read as either positive or negative. In a separate, collaborative investigation, Dr. Suresh K. Mittal (Purdue University) tested the permissiveness of pig fetal kidney and lung cells to human wtAD-5 and human Ad-5 with E1 and E3 regions deleted (similar to SCH 58500).

Single-dose studies.
Silastic catheters were surgically implanted for iv, ia, and ip dosing. The catheters were tunneled subcutaneously to exit at a site on the dorsal midline and protected by a foam-padded, customized aluminum jacket. Intravenous dosing was done by infusion into the jugular vein by a catheter directed towards the anterior vena cava or into the portal vein. Intra-arterial dosing was done by infusion into the common hepatic artery or internal carotid artery. Insertion of the catheters occurred at least 5 days prior to dosing. The rate of infusion was usually about 5 ml/min to deliver 11 x 1011 particles/min.

In Study 3, one animal was dosed via hepatic artery at a rate of 1 ml/min in addition to others dosed at a rate of 5 ml/min (9.5 x 1011 particles/ml). The intended dose in Study 3 was 47.5 x 1011 particles/kg, but because of acute onset of severe clinical signs, dosing had to be terminated after 5 min (6.4 x 1011 particles/kg) or 7 min (12.7 x 1011 particles/kg) in 2 pigs dosed at 5 ml/min, and, after 129 min (37.9 x 1011 particles/kg), in a third pig dosed at 1 ml/min. The highest dose attained was given at the 1 ml/kg rate. All pigs were euthanized on the day after dosing, as per the protocol.

Repeated-dose studies.
The repeated-dose studies were conducted by the ip and iv dose routes with female and castrated male pigs. Doses were delivered via surgically implanted catheters as described for single-dose studies. In the ip study, groups of 2 male and 3 female pigs were dosed for one or two 14-day dosing periods, separated by a 14-day dose-free period (Study 13). Two dose levels of 0.023 x and 2.2 x 1011 particles/kg/day were tested. The pigs were dosed at a rate of approximately 1 ml/kg/min and concentration of 2.2 x 1011 particles/ml. A similar group, serving as a control, was dosed for 2 dosing periods with vehicle. All pigs were euthanized on the day after completion of 1 or 2 dosing periods.

In the iv study, groups of 2 male and 3 female pigs were dosed by infusion via an implanted catheter in the portal vein for one or two 5-day dosing periods, separated by an 11-day observation period (Study 14). Two dose levels of 0.045 x and 4.5 x 1011 particles/kg/day were tested. The pigs were dosed at a rate of 5 ml/kg at a concentration of 2.39 x 1011 particles/ml. A similar group, serving as a control, was dosed for 2 dosing periods with vehicle. All pigs were euthanized on the day after completion of 1 or 2 dosing periods.

Cardiovascular/hemodynamic response.
The abrupt onset of clinical signs suggestive of an acute cardiovascular/hemodynamic response in a hepatic artery infusion study (Study 3) prompted a detailed evaluation of these potential effects in 2 subsequent studies. Cardiovascular, hemodynamic, and respiratory parameters were determined in anesthetized female pigs dosed intra-arterially into the hepatic (Study 8) or carotid (Study 9) arteries and intravenously into the jugular vein (Study 9). The pigs in Study 9 were initially anesthetized with a mixture of ketamine HCl (24 mg/kg), atropine (0.04 mg/kg), butorphanol (0.55 mg/kg), and xylazine (1 mg/kg) given intramuscularly and maintained in anesthesia with isoflurane inhalant anesthetic (in Study 8 atropine and telazol were used). The intended dose levels were 47.5 x 1011 particles/kg in Study 8 and 11 x 1011 particles/kg in Study 9. An infusion rate of 5 ml/min was used in both studies with concentrations of 9.5 x 1011 particles/ml in Study 8 and 2.25 x 1011 particles/ml in Study 9. The dose levels were set high so as to maximize the potential for causing changes, but could not be achieved by any of the dose routes because of a significant hemodynamic response. The actual doses administered were 15 or 16 x 1011 particles/kg (47.5 x 1011 particles/min for 5 and 6 min into the hepatic artery) and 3.3 x to 5.6 x 1011 particles/kg (11 x 1011 particles/min for 12 to 19 min into the carotid artery or intravenously) in Studies 8 and 9, respectively.

Measurements included heart and respiratory rates; electrocardiogram (lead II) tracings; pulmonary (PAP), systolic (SAP), diastolic (DAP), and mean (MAP) arterial pressures; left ventricular (LVP) and central venous (CVP) pressures; and cardiac output (CO). Blood gas parameters, including PO2, PCO2, TCO2 and HCO3-, were determined. A catheter sheath entering the left femoral artery and ending at the descending aorta was used for directly monitoring arterial pressures and for collection of arterial blood for the blood-gas measurements. A second catheter was directed to the left ventricle to monitor LVP. A Swan-Ganz catheter was inserted into the left femoral vein ending at a pulmonary artery, for monitoring of PAP, CVP, and CO. Hematology, serum chemistry, coagulation, and antibody assays were also performed. Blood for the determination of these parameters was collected from the right jugular vein via a catheter.

Sc and id studies.
The sc and id studies (Studies 10, 11, and 12) were designed to identify local and systemic effects following various dosing regimens, as indicated in Table 1Go. Studies 11 and 12 evaluated single or repeated sc or id injections. The lateral neck area was used as the site for sc (0.2 and 1 ml) dosing; id dosing (0.1 ml) was administered either into the pinna (Study 11) or along the dorsal midline. Repeated id and sc doses were injected at separate, adjacent sites except in Study 12, where 12 sc injection sites were sequentially used for a total of 14 to 28 injections. Study 10 evaluated 2 different formulations of SCH 58500 administered sc and id, after iv exposure to SCH 58500. Comparative iv (1 ml/kg) dosing in Study 10 was done into an ear vein. The concentration of SCH 58500 in these studies was 2.2 x 1011 particles/ml. The same pigs were dosed with vehicle at a contralateral site. The pigs were euthanized 1 to 14 days after their last dose.

Gonadal expression study.
The potential to induce gonadal expression of SCH58500-encoded p53 sequences in mature (8–15-month-old) male and female pigs was determined by giving a single intravenous dose into a peripheral ear vein of 2.2 x 1011 particles/kg (Study 7). Samples of testes and ovaries were tested for the presence of SCH 58500-encoded p53 sequences by RT-PCR and PCR.

SCH 58500 spiked with wtAd-5.
Two groups of 5 female pigs each were dosed, iv, into the jugular vein with 2.25 x 1011 particles/kg of SCH 58500 that was spiked with either 104 or 107 pfu of wild-type human adenovirus-5 (wtAD-5) (Study 6). One pig per group was euthanized on 1, 3, and 7 days after dosing and 2 pigs per group on 14 days after dosing. A control group was dosed with SCH 58500 vehicle that was also spiked with 107-pfu wtAD-5. The purpose of this study was to determine if the presence of a replication-competent wtAD-5 would adversely affect the response to SCH 58500.

In vitro hemagglutination assays.
The hemagglutination potential of adenovirus type 5, the parental strain of SCH 58500, was tested with erythrocytes from mice (Crl:CD-1® [ICR]BR VAF/PlusTM), Sprague-Dawley-derived rats (CRl:CD®BR VAF/PlusTM and BUF/NHsd), Yorkshire pigs, and Rhesus monkeys. As positive controls, erythrocytes from Sprague-Dawley rats (which display hemagglutination upon exposure to hAd5) and Yorkshire pigs sero-positive for anti-SCH 58500 antibodies were used. Rhesus monkey, a species known not to induce hemagglutination by adenovirus-5, and Yorkshire pig erythrocytes negative for anti-SCH 58500 antibodies were used as negative controls. Hemagglutination was measured by the amount of adenovirus required to produce the virus-erythrocyte matrix. Wild-type adenovirus-5 (1 x 109 pfu/l) was obtained from Advanced Biotechnologies, Columbia, MD. The assay was conducted based on the microtiter-plate method. Approximately 2 x 105 washed erythrocytes from each of the test species were placed in microtiter wells and exposed to SCH 58500 at a high concentration of {approx}1 x 108 pfu/ml and 11 serial 2-fold dilutions (final dilution of 1:2048). They were incubated at 37°C for the same amount of time as required for concurrent vehicle controls to form a distinct erythrocyte aggregate (button) at the bottom of the wells. Wells that showed a button were considered hemagglutination positive, wells with less defined or small buttons were considered partially positive, and wells that contained no distinct buttons were considered negative.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The response to SCH 58500 in single- and repeated-dose toxicity studies is summarized in the text below and, for selected studies, in Table 2Go.


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TABLE 2 High-Dose Observations in Single- and Repeated-Dose Toxicity Studies Lowest toxic dose tested membranes, lethargy, ataxia, tremor, inappetence. breathing, staggering, ataxia, fluid accumulation, prostrate, tremors,
 
Host permissiveness study (Study 1, Table 1Go).
An intra-arterial dose of 2.2 x 1011 particles/kg of wtAD-5 was well tolerated, causing no changes in clinical signs, clinical laboratory measurements or microscopic lesions. Viral cultures from liver, spleen, and thoracic lymph nodes collected the day following dosing demonstrated a wtAD-5-induced cytopathic effect. No cytopathic effect was observed at later test intervals. In the independent in vitro assays, human wtAD-5 replication occurred in fetal kidney and lung cells from pigs (data not shown).

Single-dose toxicology/toxicokinetic studies.
Significant signs of toxicity were observed in Study 3 (Table 1Go) in which the highest dose level of SCH 58500 was tested. In this study infusion of 6.4 x to 37.9 x 1011 particles/kg caused severe signs of toxicity that limited the deliverable dose. The signs included vomiting, collapse, prostration, rapid or labored shallow breathing, ataxia, pale/cyanotic mucous membranes, tremors, lethargy, and inappetence. Signs of toxicity were first observed at about 5 min (47.5 x 1011 particles/min infusion) or 2 h (9.5 x 1011 particles/min) following initiation of infusion; recovery was evident at 24 h after dosing. A mild decrease in lymphocyte counts occurred 30 min after the initiation of infusion. At 6 h, lymphocyte counts were moderately decreased, platelet counts mildly decreased, and marked increases in mature and immature (band) neutrophils were observed. These findings are suggestive of an initial stress response, followed by inflammation.

Mildly increased neutrophil counts, circulating band neutrophils, and moderately decreased platelet counts were present 24 h after dosing. Mild increases in aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), and alkaline phosphatase (ALP) values, as well as urea nitrogen (BUN) and creatinine values, were observed but were not associated with microscopic changes in the kidney or liver at 24 h postdose. Microscopic changes were limited to the lung and lymph nodes and included pinpoint discoloration on the lungs correlating with extravasated red blood cells and enlarged, purple, mottled tracheobronchial and/or mediastinal lymph nodes correlating with congestion, with or without hemorrhage, and hemosiderin deposition. There were no effects on urinalysis parameters or organ weights in any of the single-dose studies.

In the other single-dose studies, dose levels as high as 2.2 x 1011 particles/kg given intra-arterially (hepatic artery; Study 2, Table 1Go) or intravenously (jugular or ear vein; Studies 4 and 7, Table 1Go, respectively) were well tolerated with no evidence of toxicity. There were no deaths in any studies that were attributed to SCH 58500. One male in Study 2 dosed with 0.022 x 1011 particles/kg was found dead 2 weeks postdose due to hepatic necrosis resulting from complications associated with catheterization of the hepatic artery. Also in Study 2, one female pig infused with 2.2 x 1011 particles/kg had seizure-like activity 11 days following dosing and was euthanized. This pig had high plasma ammonia levels and metabolic acidosis that were not associated with SCH 58500. These effects were not temporally associated with dosing and did not occur in other studies at this dose. While an association with SCH 58500 cannot be definitively ruled out, it was considered unlikely, based on an overall assessment of the compound.

In two of the single-dose studies (Studies 2 and 5, ia and ip, respectively, at 2.2 x 1011 particles/kg), anti-SCH 58500 antibodies and serum neutralizing factors increased after dosing. Infectious activity (plaque-forming units) was present in the sera of some of the high-dose pigs dosed by the ia (Study 3) and iv (Study 4) routes, most commonly at 5 to 15 min postdose, but was not observed in pigs dosed by the ip route (Study 5). SCH 58500-encoded p53 DNA was detected in sera of pigs given the high dose levels by the ia (Studies 2 and 3) and iv (Study 4) routes but not in those dosed ip (Study 5). SCH 58500-encoded p53 mRNA was observed in spleen, liver, lung, thoracic lymph node, and mesentery/omental tissue samples taken primarily on the day following dosing but also up to 7 days following ia and ip dosing (Studies 2, 3, and 5) and for up to 2 weeks in the lungs and spleen after iv dosing with 0.75 x 1011 particles/kg. SCH 58500-encoded p53 DNA was detected in spleen, colon, mesentery/omental tissue, and liver when measured 2 and 3 days following ip dosing at 2.2 x 1011 particles/kg in Study 5.

When measured in Study 4, SCH 58500 did not induce hemagglutination of erythrocytes in any of the pigs in this study.

Repeated-dose study, ip route.
Transient clinical signs of toxicity were observed in pigs dosed ip with 2.2 x 1011 particles/kg/day for two 14-day dose periods (Study 13). These signs included lethargy, inappetence, and emesis starting at about 4 to 6 h postdose. The signs were initially observed only in males following their first dose. In addition to these signs, males exhibited a wagging of their hindquarters and assumption of a sitting position, or became prostrate during dosing and had rapid shallow breathing and tremors at 3 to 8 h postdose. In addition, at about 5 to 6 h postdose, 1 or 2 males showed distended abdomens and rales, erythema of the abdomen and feet, and, at 24 h postdose, rectal prolapse. The females responded similarly but not until after the second dose, and overall, their response was less severe than that of the males. During the 14-day dosing period, the incidence and severity of signs in males and females decreased with continued dosing beyond the second day, and were not usually observed with dosing on subsequent days. Those pigs dosed for a second 14-day dosing period after a 14-day rest interval had comparable, but less severe, signs. Again, signs were generally associated with the first day of reexposure and were typically less common and/or severe in females. All pigs appeared normal between the first and second dosing periods and after the second 14-day dosing period prior to euthanasia.

Male and female pigs dosed with 2.2 x 1011 particles/kg/day had minimally (22 to 29%) to moderately (41 to 91%) higher fibrinogen values when compared to the controls, beginning on day 2 and continuing through the dosing periods. Increased fibrinogen is suggestive of inflammation. Serum total protein and globulin levels were increased and albumin:globulin ratios decreased. Globulin concentrations were greater at the end of the second dosing period than at the end of the first dosing period. There were no other changes in serum chemistry parameters.

Histopathologic observations attributable to SCH 58500 were limited to an increased incidence over controls of generalized peritoneal inflammation involving abdominal viscera (excess fluid, serosal discoloration and surface irregularity, and adhesions) in pigs dosed with 2.2 x 1011 particles/kg/day. There were no SCH 58500-related changes in body weights, hematology, or urinalysis measurements at the high-dose level. No signs of toxicity were observed at the low dose (0.023 x 1011 particles/kg/day).

Anti-SCH 58500 antibody levels and serum neutralizing factor activity were increased in all pigs over the course of dosing, in a dose-related manner. Pigs in both dose-level groups had higher antibody levels following the second cycle of dosing. SCH 58500-encoded p53 DNA sequences were detected in a few serum samples collected 10 to 30 min following dosing with 2.2 x 1011 particles/kg/day. SCH 58500-encoded p53 mRNA sequences were not detected in the tissues sampled (liver, spleen, and mesentery/omentum).

Repeated-dose study, iv route (Study 14, Table 1Go).
There were no SCH 58500-related effects on hematology, serum chemistry, coagulation, and urinalysis parameters in pigs dosed iv (portal vein) for one or two 5-day intervals with 0.045 and 4.5 x 1011 particles/kg/day. Body weights were not affected by dosing and there were no unusual necropsy observations or microscopic lesions. Signs of toxicity were limited to clinical observations in pigs given the high-dose level. As with ip dosing, almost all signs were observed during or immediately following the initial dose in 60% of the males and 20% of the females. They included lethargy, rapid breathing, staggering, ataxia, and prostration. The pigs appeared normal by 1 h after dosing and for the remainder of the day, with minor exceptions. One female was lethargic and not eating 5 h after the first dose and another female was lethargic and staggering during the time the fifth dose was administered.

Anti-SCH 58500 antibodies increased in most pigs dosed with SCH 58500 during the dosing period. High-positive, serum-neutralizing factors developed in all SCH 58500-dosed pigs and 3 control pigs. Quantifiable SCH 58500-encoded p53 DNA was detected in the sera of all high dose-group pigs on the first or second dose day but in none of the low dose-group pigs. Encoded p53 DNA was observed in fewer pigs as the study progressed. Plaque-forming units were detected in samples of both low- and high-dose pigs during the first 2 days of dosing only. SCH 58500-encoded p53 DNA was detected in liver tissue from all pigs tested at the end of the first and second dosing periods.

Cardiovascular/hemodynamic response.
Infusion of 3.5 x 1011 particles/kg (47.5 x 1011 particles/min) and above into the hepatic artery, internal carotid artery, or jugular vein caused severe hemodynamic effects in anesthetized pigs (Studies 8 and 9). Starting at 2 to 10 min after the initiation of infusion, we observed sporadic premature ventricular contractions (PVCs), increased PAP (~50–400%), decreased SAP, DAP, and MAP (~15–800%), decreased LVP (~5–85%), increased CVP (up to 1800%), and reduced cardiac output (~60–100%). Heart and respiratory rates were erratic.

These changes, indicative of systemic hypotension and pulmonary hypertension, resulted in poor circulation, insufficient oxygenation, and significant hypoxemia (75% decrease in pO2 about 10 to 30 min following infusion). Respiratory rates decreased about 50%.

The time course of changes was similar among pigs regardless of the dose route. The responses were similar at the range of 3.5 x to 16 x 1011 particles/kg. PAP, but no other hemodynamic parameter, returned to baseline values 4 to 5 min after termination of dosing, following delivery of 15–16 x 1011 particles/kg. All pigs, dosed either iv or ia via the carotid artery, were euthanized due to the severity of hemodynamic changes following infusion of doses ranging from 3.3 to 5.6 x 1011 particles/kg. Examples of the cardiovascular and hemodynamic changes are given in Figure 1Go and Table 3Go.



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FIG. 1. Hemodynamic response of pigs dosed by infusion into the internal carotid artery with SCH 58500 at a rate of 11.25 x 1011 particles/min. Infusions were stopped at an approximate mean time of 17 min. Means of heart rate (HR), systolic (SAP), diastolic (DAP), and mean (MAP) arterial pressures, left ventricular pressure (LVP), and central venous pressure (CVP) of 2 pigs are plotted.

 

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TABLE 3 Arterial Blood Gases in Pigs Dosed Intra-arterially with SCH 58500
 
Approximately 5 min after initiation of dosing with 47.5 x 1011 particles/min, progressive decreases in total white blood cell, neutrophil, and platelet counts occurred. These decreases became marked by 30 min after cessation of infusion (Table 4Go). Mild lymphopenia was also observed. Plasma fibrinogen values were markedly reduced at 30 min post infusion, and no fibrin-degradation products were present. PT and APTT values were decreased. The data suggest a possible effect on coagulation/fibrinolysis balance, but are not indicative of disseminated intravascular coagulation, which classically presents as decreased platelets and fibrinogen with increased FDP's, PT, and APTT. These changes were followed by decreases in erythrocyte and protein parameters as well as decreases in a variety of serum components secondary to hemodilution.


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TABLE 4 Selected Clinical Laboratory Values of Pigs Dosed Intra-arterially with SCH 58500
 
Subcutaneous and intradermal studies (Studies 10–12, Table 1Go).
SCH 58500 was generally well tolerated at the sites of sc, id, and iv injections. A summary of observations is given in Table 5Go. A localized minimal to mild chronic inflammatory response was observed after id dosing. The local response to dosing was not affected by multiple injections at different sites. However, the severity and persistence of the inflammatory response were dose-related and were greater at the sites receiving multiple as compared to single injections. The immune response to SCH 58500 observed after a single dose increased following multiple (5 to 28) doses. The development of antibody levels was similar following continuous or multicycle (three 5-day dosing periods) dosing. No SCH 58500-encoded p53 mRNA sequences were detected in the liver, lung, or spleen samples collected 1 to 14 days following dosing (Study 10). However, SCH 58500-encoded p53 mRNA sequences were detected in one of the assayed intradermal injection sites. In Study 10, the responses to each formulation tested by the sc, id, and iv routes were generally indistinguishable. There was no indication of enhanced reactogenicity at id/sc sites subsequent to initial iv exposure.


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TABLE 5 Summary of Studies in Yorkshire Pigs Dosed with SCH 58500 by the Subcutaneous and Intradermal Routes
 
SCH 58500 spiked with wtAD-5 (Study 6).
The spiking of SCH 58500 (2.2 x 1011 particles/kg) with wtAD-5 (104 and 107 pfu) had no effect on the various observations and measurements made during the study. There was no histopathologic indication of formation of intranuclear inclusion bodies (indicative of replication of wild-type human adenovirus) in any tissue. All pigs had at least a 2-fold increase in anti-SCH 58500 antibodies and positive results from SNF. Serum SCH 58500 (infectious titer and/or p53 DNA) was detectable only within 10 min postdose. No encoded mRNA sequences were detected in liver, lung, spleen, or thoracic lymph node.

Gonadal expression study (Study 7).
No SCH 58500-encoded p53 sequences were detected in gonadal tissues of sexually mature male and female pigs collected 21 days following a single iv dose. No SCH 58500 encoded p53 RNA was detected in sperm pellet samples. There were no clinical signs of toxicity and no microscopic lesions in the testes or ovaries.

In vitro hemagglutination assays.
WtAD-5 did not induce hemagglutination in mouse or negative-control monkey erythrocytes. Hemagglutination was observed in Sprague-Dawley rat erythrocytes and anti-SCH 58500 antibody sero-positive Yorkshire pig erythrocytes (complete hemagglutination ranging from 1:5 to 1:10 dilutions and incomplete hemagglutination from 1:20 to 1:40 dilutions). WtAD-5 did not induce hemagglutination in erythrocytes from any of the Buffalo rats tested.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pig has previously been reported (Betts et al., 1962Go; Torres et al., 1996Go) to be a permissive host for adenovirus replication. Observations of a wtAD-5-induced cytopathic effect in the liver, spleen, and thoracic lymph nodes occurred only on the day following dosing with wtAD-5 (Study 1). The observation is not considered conclusive evidence for adenovirus replication in porcine tissue but does suggest that the Yorkshire pig is a permissive host. Data more supportive of the appropriateness of the pig as a test species comes from the in vitro assays, which indicate that porcine fetal kidney and lung cells are permissive to wtAD-5 and human Ad-5 replication (data not shown). The tissue expression data in pigs used in the toxicity studies confirmed that tissue encoding of p53 can occur and that the pig is an appropriate test species. These data allow evaluation of potential toxicity during coinfection with wtAD-5 and SCH58500, as well as replication-competent adenoviruses present in SCH 58500. No adverse effects were seen in pigs at doses used to study wtAD-5 and SCH 58500.

The minimal detection of serum levels of SCH 58500 and SCH 58500-encoded p53 RNA sequences was likely the result of rapid clearance from the sera of immune pigs. Time-related absence of detectable pfu on successive days of dosing in high-dose pigs suggests that SCH 58500 was being "cleared" from the sera in a progressively more efficient fashion. Clearance was likely mediated by the development of anti-SCH 58500 antibodies and/or neutralizing factors. If enhanced clearance is observed in clinical trials, overall efficacy in humans may be mitigated.

SCH 58500 was well tolerated in pigs given single and repeated-dose regimens by several dose routes. Single iv, ia, and ip doses of 2.2 x 1011 particles/kg caused no toxicity. Repeated doses of 2.2 x 1011 particles/kg/day given ip and 4.5 x 1011 particles/kg/day injected into the hepatic vein caused clinical signs of toxicity that disappeared with continued dosing. The reason for the differences in clinical signs observed in response to the ip dose of 2.2 x 1011 particles/kg between Studies 5 and 13 (Table 2Go) is not known, although the animals in the repeated-dose study (Study 13) weighed less than those in the single-dose study (Study 5). The severity and appearance of clinical signs was dose-related and related to the rate of infusion. Although the number of pigs dosed was limited, the threshold dose for causing significant cardiovascular and hemodynamic changes is about 6.4 x 1011 particles/kg.

Observations in response to high doses are suggestive of an immune-mediated response. The rapid appearance of vomiting, cyanosis, decreases in platelets, hypotension and cardiovascular collapse, erythema of the abdomen and feet, and difficulty in breathing are suggestive of a response in which the changes may be the result of released inflammatory mediators. Depending on dose, the clinical observations were reversible in the pigs, possibly due to rapid clearance and development of SNF.

Decreased lymphocyte and platelet counts in the hepatic artery infusion study (Study 3) suggest a stress response, possibly followed by a SCH 58500-induced inflammatory response, with demargination of neutrophils or early release of band neutrophils. Decreased platelets are consistent with vasculitis or local consumption. Signs of pooling or destruction of red blood cells in the lung and liver may be related to hemodynamic effects associated with the response to SCH 58500 rather than a direct effect of the test substance.

The increases in serum concentrations of liver enzymes and increases in urea nitrogen and creatinine levels observed in Study 3 were not accompanied by changes in the morphology of the liver and kidney. Increased BUN and creatinine were probably prerenal (hypovolemia). The possible cause of the liver enzyme changes is not known. They may be a secondary or tertiary result of a local immune reaction in the liver or a consequence of the hemodynamic disruption associated with high doses of SCH 58500. However, hepatic toxicity has been observed in murine cancer models exposed to high concentrations of recombinant adenovirus (Nielsen et al., 1998Go), and a direct, mild effect of SCH 58500 on the liver cannot be ruled out.

Relatively high dose levels of SCH 58500 caused severe systemic hypotension and pulmonary hypertension that resulted in poor circulation, insufficient oxygenation, significant hypoxemia, and minimal to mild metabolic acidosis. These hemodynamic and cardiac effects were associated with decreases in white blood cells and platelets and, as suggested above, may have indirectly led to the serum chemistry changes implicating the liver and kidney as target organs. The hemodynamic effects of SCH 58500 were dose-related and do not appear to occur below intra-arterial doses of 2.2 x 1011 particles/kg.

SCH 58500 was generally well tolerated at both dose levels tested in the repeated-dose iv (hepatic vein) study (Study 14). Signs of toxicity were limited to transient clinical signs during or immediately following dosing with 4.5 x 1011 particles/kg/day primarily on the first 2 dose days only and also after a nondosing interval. These observations correlated with detection of SCH 58500 in sera (plaque assay) only on these dose days and high positive SNF levels in all pigs. The time-related decrease in the incidence of p53 DNA in sera samples and the absence of detectable pfu in high-dose pigs indicate that SCH 58500 was being cleared from sera in a progressively more efficient fashion, which likely accounts for the increase in tolerance with repeated dosing.

Initial clinical signs of toxicity appeared to be more severe and continued for a longer period in the repeated-dose ip study (Study 13) than in the repeated-dose iv study. In the repeated-dose ip study, higher fibrinogen values are considered caused by an immune or inflammatory response related to the highest dose levels tested. An increase in globulin and anti-SCH 58500 antibody levels between the end of the first and second dosing cycles in pigs dosed ip suggest an anamnestic antibody (gamma globulin) response after the second cycle. These changes are compatible with antibody production due to a humoral immune response to high doses of SCH 58500.

In the local toxicity studies, multicycle sc or id dosing resulted in increased levels of anti-SCH 58500 antibodies relative to baseline. However, there was no histopathologic evidence of increased reactogenicity or a delayed-type hypersensitivity response, perhaps because of the increased development of anti-SCH 58500 neutralizing antibody levels. SCH 58500-encoded mRNA was not detected in visceral organs and rarely in intradermal sites, suggesting a rapid clearance.

Adenoviruses are known to induce hemagglutination of mammalian erythrocytes in in vitro studies. Hemagglutination is species-specific and specificity is determined by the presence of erythrocyte surface receptors that interact with the adenovirus fiber protein. Hemagglutination data is usually used for classification of viruses. The in vivo biological significance of the in vitro hemagglutination assays with erythrocytes taken from sero-positive Yorkshire pigs from the same source used in the toxicity studies is not known.

The pigs used in these studies had antibodies that bound to SCH 58500 and, in most pigs, neutralized the vector. In the high-dose animals, some serum samples were positive in the PCR assay and SCH 58500-encoded p53 mRNA was detected in the spleen and liver on the day following dosing but not at later times. Only a small section of each organ is tested. Therefore a lack of detection of specific mRNA does not necessarily rule out its presence. SCH 58500 expression, i.e., p53 RNA from SCH 58500, appears to be present (in lungs and spleens) of immune animals for as long as 2 weeks after an iv dose of 0.75 x 1011 particles/kg, although in most animals, circulating virus is only detectable for up to 10 min. One pig in the high-dose group (Study 4) that was negative for the virus in the plaque assay was a high positive in the neutralizing assay. However, SCH 58500-encoded mRNA could be detected in the tissues from this animal, so the antibody did not preclude expression of the gene.

These studies, together with those described in a companion manuscript (Morrissey et al., 2002Go) formed the basis for initial clinical trials. Starting doses for intratumoral (head and neck), ovarium, and hepatic oncology indications were selected based on no-effect doses from these studies.


    ACKNOWLEDGMENTS
 
A large number of individuals contributed to successful completion of the studies which formed the basis of this manuscript. We would like to thank Drs. Wai Nang, Dinesh Sinha, Larry Mortin, Bob Johnson, Margaretann Halleck, Janet Petruska, Narendra Kishnani, Ron Bordens, Ted Schmahai, Bob Veneziale, Tom Haushalter, George Mandakas, Andy Kiorpes, Andras Fabray, and G. Kaminska-McNamara, Mr. C. Reilly, and Ms. LaMantia for invaluable assistance. We appreciate the expert assistance of A. Thotathuchery-Sanders in preparation of this manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (973) 940-4211. E-mail: richard.morrissey{at}spcorp.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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