Synagis
Россия
  • Россия
  • Украина

Synagis - Scientific Information

Manufacture: AbbVie
Country: Canada
Condition: Respiratory Syncytial Virus
Class: Immune globulins
Form: Liquid solution, Intramuscular (IM), Powder
Ingredients: palivizumab, glycine, histidine, mannitol

Pharmaceutical information

Drug Substance

Proper name:palivizumab
Structural formula:



Product
characteristics:
Palivizumab is produced by recombinant DNA technology in a mammalian cell (NSO) suspension culture. The anti-respiratory syncytial virus (RSV) antibody is purified by affinity and ion-exchange chromatography steps. The purification process includes specific viral inactivation and removal procedures. Palivizumab is a humanized IgG1 monoclonal antibody directed to an epitope in the A antigenic site of the fusion protein of RSV. Palivizumab specifically binds with high affinity (Kd = 0.96 nM) to the F protein of RSV. Palivizumab is a composite of (95%) human and (5%) murine amino acid sequences. The antibody contains about 1 to 2% carbohydrate by weight which is composed of N-acetyl-glucosamine, mannose, fructose, and galactose.

This humanized monoclonal antibody is composed of two heavy chains (50.6 kDa each) and two light chains (27.6 kDa each), has a molecular weight of approximately 148,000 Daltons and an isoelectric point of greater than 9.0.

Clinical Trials

Study Demographics and Trial Design

The safety and efficacy of SYNAGIS (palivizumab) were assessed in a randomized, double-blind, placebo-controlled trial (IMpact-RSV Trial) of respiratory syncytial virus (RSV) disease prophylaxis among children with premature birth and children with bronchopulmonary dysplasia (BPD), and in a randomized, double-blind, placebo-controlled trial of RSV disease prophylaxis among children with hemodynamically significant congenital heart disease (CHD) (Study MI-CP048). Additional clinical studies conducted following the initial approval of SYNAGIS have provided further data on the safety and effectiveness of SYNAGIS prophylaxis for the prevention of RSV related diseases among the similar pediatric populations.

Study Results

Study MI-CP018 (IMpact-RSV Trial)

Study MI-CP018, a randomized (two to one, palivizumab to placebo), multinational, double blind, placebo-controlled clinical trial conducted during the 1996 to 1997 RSV season in 1,502 pediatric subjects with prematurity (< 35 weeks of gestation) or bronchopulmonary dysplasia who received 5 monthly intramuscular injections of 15 mg/kg palivizumab or placebo and who were followed for an additional 150 days (30 days after the last injection), conducted at 139 centres in the United States, Canada and the United Kingdom, studied patients ≤ 24 months of age with BPD and patients with premature birth ≤ 35 weeks gestation who were ≤ 6 months of age at study entry. Patients with uncorrected CHD were excluded from enrolment. In this trial, 500 patients were randomized to receive five monthly placebo injections and 1002 patients were randomized to receive five monthly injections of 15 mg/kg of SYNAGIS lyophilized powder formulation. Subjects were randomized into the study and were followed for safety and efficacy. Ninety-nine percent of all subjects completed the study and 93% received all five injections. The primary endpoint was the incidence of RSV hospitalization.

RSV hospitalizations occurred among 53 of 500 (10.6%) patients in the placebo group and 48 of 1002 (4.8%) patients in the SYNAGIS group, a 55% reduction (p < 0.001). The reduction of RSV hospitalization was observed both in patients enrolled with a diagnosis of BPD (34/266 [12.8%] placebo vs 39/496 [7.9%] SYNAGIS) and patients enrolled with a diagnosis of prematurity without BPD (19/234 [8.1%] placebo vs 9/506 [1.8%] SYNAGIS). The reduction of RSV hospitalization was observed throughout the course of the RSV season.

Among secondary endpoints, the incidence of intensive care unit (ICU) admission during hospitalization for RSV infection was lower among subjects receiving SYNAGIS (1.3%) than among those receiving placebo (3.0%), but there was no difference in the mean duration of ICU care between the two groups for patients requiring ICU care. Overall, the data do not suggest that RSV illness was less severe among patients who received SYNAGIS and who required hospitalization due to RSV infection than among placebo patients who required hospitalization due to RSV infection. SYNAGIS did not alter the incidence and mean duration of hospitalization for non-RSV respiratory illness or the incidence of otitis media.

Study MI-CP110

Study MI-CP110, conducted at 347 centers in the North America, European Union and 10 other countries, studied patients less than or equal to 24 months of age with CLDP and patients with premature birth (less than or equal to 35 weeks gestation) who were less than or equal to 6 months of age at study entry. Patients with hemodynamically significant congenital heart disease were excluded from enrollment in this study and were studied in a separate study. In this trial, patients were randomized to receive 5 monthly injections of 15mg/kg of SYNAGIS solution for injection (N=3306) used as active control for an investigational monoclonal antibody (N=3329). Subjects were followed for safety and efficacy for 150 days. Ninety-eight percent of all subjects receiving SYNAGIS completed the study and 97% received all five injections. The primary endpoint was the incidence of RSV hospitalization.

RSV hospitalizations occurred among 62 of 3306 (1.9%) patients in the SYNAGIS group. The RSV hospitalization rate observed in patients enrolled with a diagnosis of CLDP was 28/723 (3.9%) and in patients enrolled with a diagnosis of prematurity without CLDP was 34/2583 (1.3%).

Study MI-CP048

Study MI-CP048, conducted at 76 centers in the United States, Canada, France, Germany, Poland, Sweden and the United Kingdom, studied patients ≤ 24 months of age with hemodynamically significant CHD. In this trial, 648 patients were randomized to receive five monthly placebo injections and 639 patients were randomized to receive five monthly injections of 15 mg/kg of SYNAGIS lyophilized powder formulation. The trial was conducted during four consecutive RSV seasons. Subjects were stratified by cardiac lesion (cyanotic vs. other) and were followed for safety and efficacy for 150 days. Ninety-six percent (96%) of all subjects completed the study and 92% received all five injections. The primary endpoint was the incidence of RSV hospitalization.

RSV hospitalizations occurred among 63 of 648 (9.7%) patients in the placebo group and 34 of 639 (5.3%) patients in the SYNAGIS group, a 45% reduction (p = 0.003). The reduction of RSV hospitalization was consistent over time, across geographic regions, across stratification by anatomic cardiac lesion (cyanotic vs. other), and within subgroups of children defined by gender, age, weight, race, and presence of RSV neutralizing antibody at entry. The secondary efficacy endpoints that showed significant reductions in the SYNAGIS group compared to placebo, included total days of RSV hospitalization (56% reduction, p = 0.003) and total RSV days with increased supplemental oxygen (73% reduction, p = 0.014).

Study MI-CP124

Study MI-CP124 conducted at 162 centers in North America, European Union and 4 other countries over two RSV seasons, studied patients less than or equal to 24 months of age with hemodynamically significant CHD. In this trial, patients were randomized to receive 5 monthly injections of 15mg/kg of SYNAGIS solution for injection (N=612) used as active control for an investigational monoclonal antibody (N=624). Subjects were stratified by cardiac lesion (cyanotic vs. other) and were followed for safety and efficacy for 150 days. Ninety-seven percent of all subjects receiving SYNAGIS completed the study and 95% received all five injections. The primary endpoint was a summary of adverse events and serious adverse events, and the secondary endpoint was the incidence of RSV hospitalization. The incidence of RSV hospitalization was 16 of 612 (2.6%) in the SYNAGIS group.

Study MI-CP026 - Reduction of Viral Load in Tracheal Aspirates

A study was conducted in children hospitalized and intubated with RSV infection to determine whether SYNAGIS reduced RSV titers in tracheal secretions; 17 children were randomized to receive a single intravenous infusion of 15 mg/kg SYNAGIS and 18 children to receive placebo. The results are presented in Table 1.

Table 1. Quantitative RSV Plaque Assay Titer (log10) Tracheal Aspirates
Placebo
(SE)
SYNAGIS
(SE)
p-value
Mean Titer at Study Entry 4.8 (0.3) 4.8 (0.3)
Decrease in Titer on Day 1 0.6 (0.2) 1.7 (0.3) 0.004
Decrease in Titer on Day 2 1.0 (0.4) 2.5 (0.3) 0.012
Decrease in Titer on Day 3 1.9 (0.7) 2.8 (0.4) 0.288
Decrease in Titer on Day 4 2.1 (0.7) 2.8 (0.5) 0.500
Decrease in Titer on Day 5 1.8 (0.7) 2.7 (0.5) 0.417

definitions: SE = standard error

SYNAGIS was found to reduce the tracheal RSV titer significantly when compared to placebo. However, despite the antiviral effect of SYNAGIS observed, no difference in the severity of RSV disease was observed in three treatment studies; days of RSV hospitalization, days of mechanical ventilation and days of hospitalization with a supplemental oxygen requirement were similar in the placebo and SYNAGIS groups.

Comparative Bioavailability Studies

Study MI-CP080

The pharmacokinetics, safety and tolerability SYNAGIS lyophilized powder and solution for injection formulations following intramuscular and intravenous dosing were evaluated in Study MI-CP080. In this study, healthy adult male and female volunteers 18 to 49 years of age were randomized to receive SYNAGIS lyophilized powder or solution for injection in a double-blind fashion as follows:

Group 1 (N = 12): 3 mg/kg, intramuscular, solution for injection at Study Days 0 and 30
Group 2 (N = 12): 3 mg/kg, intramuscular, lyophilized powder at Study Days 0 and 30
Group 3 (N = 12): 15 mg/kg, intravenous, solution for injection at Study Day 0
Group 4 (N = 12): 15 mg/kg, intravenous, lyophilized powder at Study Day 0

The intramuscular administration of SYNAGIS was limited to a dose of 3 mg/kg due to the volume of injection limitations in an adult volunteer; injections were given twice, once on Day 0 and once on Day 30. The intravenous administration was given once on Day 0.

Pharmacokinetic parameters for SYNAGIS lyophilized and solution for injection formulations following 3 mg/kg intramuscular administration and 15 mg/kg intravenous administration are presented in Table 2. These results support the similarity of pharmacokinetics between the solution for injection and lyophilized powder formulations of SYNAGIS.

Table 2. Summary of Pharmacokinetic Parameters Following Administration of SYNAGIS® in Healthy Adult Male and Female Volunteers 18 to 49 Years of Age (Study MI-CP080)
Parameter 3 mg/kg IM 15 mg/kg IV
SYNAGIS
Solution for
Injection
Formulation
N=12
Arithmetic Mean
(SE)
SYNAGIS
Lyophilized
Powder
Formulation
N=12
Arithmetic Mean
(SE)
SYNAGIS
Solution for
Injection
Formulation
N=12
Arithmetic Mean
(SE)
SYNAGIS
Lyophilized
Powder
Formulation
N=12
Arithmetic Mean
(SE)
AUC0-∞
(mcg•day/mL)
890 (111.5) 844 (119.9) 6673 (749.2) 6310 (413.2)a
AUC0-30
(mcg•day/mL)
569 (56.5) 511 (44.1) 4240 (335.1) 4390 (229.2)
Cmax
(mcg/mL)
32.6 (2.35) 29.6 (2.84) 502.5 (35.89) 585.0 (32.4)a
Tmax
(days)
3.063 (0.2603) 3.889 (0.5944) 0.161 (0.0503) 0.094 (0.0466)
t½
(days)
19.8 (3.38) 20.1 (3.28) 20.6 (2.23) 18.3 (1.93)

Detailed Pharmacology

Pre-Clinical

In vitro Studies of Binding RSV

In vitro studies demonstrated the potent binding activity of palivizumab to the RSV F protein. The in vivo studies performed are listed in Table 3.

Table 3. Summary of In Vitro Studies Performed with Palivizumab
Property Experiment
Affinity ELISA
Kinetic analysis
Neutralization of RSV Microneutralization
Plaque reduction
Neutralization of clinical isolates

The studies demonstrate that the Kd of palivizumab is approximately 1 nM, which is equal to or better than that of an isotype matched chimeric version of the parent MAb. Neutralization studies indicate that palivizumab is at least equivalent to the chimeric version of MAb 1129 in a microneutralization assay and that palivizumab effectively neutralizes both A and B laboratory subtypes.

To confirm the expected efficacy in the clinical setting, a diverse panel of clinical isolates of both A and B subtype RSV from European and North American sites were evaluated.

Palivizumab was tested against a panel of 57 clinical isolates of RSV, of both the A and B subtypes, isolated between 1987 and 1993 from different geographic areas of the United States. The origin and subtype of these isolates is presented in Table 4. The microneutralization assay was used with the concentration of antibody fixed (400 ng/mL) while the virus was serially diluted. Polyclonal RespiGam served as a positive control and a non-neutralizing human antibody developed at MedImmune as a negative control. All 57 of the North American clinical isolates tested were neutralized by palivizumab.

Table 4. Neutralization of Clinical Isolates by Palivizumab (North American Data)
Source RSV-A RSV-B Total
Rochester 6 4 10
Huston 9 7 16
St.Louis 2 5 7
Nashville 5 1 6
Long Beach 4 1 5
Denver 3 2 5
Other 5 3 5
Total 34 23 57

In addition to the isolates obtained from the North American studies, twenty laboratory adapted RSV isolates were obtained from Europe, as presented in Table 5.

Table 5. Neutralization of Clinical Isolates by Palivizumab (European Data)
Source RSV-A RSV-B Total
UK 10 4 14
Spain 3 1 4
Sweden 1 1 2
Total 14 6 20

All 20 isolates were neutralized by palivizumab.

These studies demonstrate that palivizumab is effective in neutralizing a range of RSV-A and RSV-B subtypes that occur clinically in North America and in Europe.

Animal

Pharmacodynamics

Cotton Rat In Vivo Activity

Respiratory Syncytial Virus bronchiolitis and interstitial pneumonia can be experimentally induced in the cotton rat, producing pathology qualitatively similar, but less severe, to that seen in the human. Three efficacy studies were conducted using the cotton rat model for RSV bronchopneumonia. The studies evaluated the reduction in pulmonary viral load and/or histopathology after either intravenous or intramuscular administration as prophylaxis or treatment of RSV.

Intravenous infusion in the treatment of RSV in cotton rats

Palivizumab was evaluated for its ability to treat an ongoing infection in cotton rats using intravenous infusion at doses of 0.63 to 10.0 mg/kg. Bovine serum albumin (BSA) and RespiGam were used as negative and positive controls, respectively, for this experiment. One day after treatment, the animals were sacrificed and the circulating human antibody and pulmonary RSV titer were determined. The results of this experiment are summarized in Table 6.

Table 6. Therapy of RSV Infection by Palivizumab in Cotton Rats
Sample Dose (mg/kg) Humanized IgG (mcg/mL) Lung RSV Titer pfu/g (mean log10 ± SE)
BSA 10 0 5.99 ± 0.1
Palivizumab 0.63 4.1 5.13 ± 0.16
Palivizumab 1.25 18.8 4.74 ± 0.08
Palivizumab 2.5 20.2 4.94 ± 0.19
Palivizumab 5 60.2 3.37 ± 0.13
Palivizumab 10 106 2.81 ± 0.27
RSV-IGIV 500 3100 < 2 ± 0

Definitions: BSA = Bovine serum albumin; RSV-IGIV = RespiGam; SE = standard error.

These results demonstrated a dose response where a 10 mg/kg palivizumab treatment resulted in circulating levels of approximately 100 mcg/mL at the time of sacrifice and a reduction in RSV titer of three orders of magnitude (three log10). Thus, palivizumab is effective in reducing viral titer when administered to RSV infected cotton rats.

Intramuscular injection in cotton rats

Intramuscular dose-ranging studies were conducted in the cotton rat model. Palivizumab or RespiGam was administered by intramuscular injection. The doses of palivizumab consisted of 5 mg/kg (body weight), 1.67 mg/kg, and 0.56 mg/kg. For comparison, polyclonal human RespiGam was administered at doses of 250 mg/kg, 50 mg/kg, and 16.7 mg/kg (Experiment I) or 250 mg/kg, 83.3 mg/kg, and 27.8 mg/kg (Experiment II). In both experiments bovine serum albumin (BSA) at 5.0 mg/kg was used as the negative control. Twenty-four hours after administration of palivizumab, the animals were bled and infected intranasally with RSV. Four days after inoculation, animals were sacrificed, and their lungs were harvested and titered in vitro. Human antibody concentration in the serum at the time of challenge was determined. Results of these experiments are presented in Table 7.

Table 7. Assessment of Potential for Palivizumab to Treat RSV (Long Strain) Infection in Cotton Rats Following a Single Intramuscular Injection (Part I)
Compound Number
of Animals
Dose
(mg/kg)
Serum [Human IgG] at Challenge
(mcg/mL)
Lung Viral Titer pfu/g
(mean log10 ± SE)
Experiment I
BSA45.005.20 ± 0.07
Palivizumab40.5624.66 ± 0.07
Palivizumab41.67112.66 ± 0.38
Palivizumab45.0302.17 ± 0.17
RSV-IGIV416.71254.48 ± 0.04
RSV-IGIV4502983.87 ± 0.11
RSV-IGIV425014502.29 ± 0.29
Experiment II
BSA45.003.75 ± 0.24
Palivizumab40.5642.15 ± 0.09
Palivizumab41.67132.08 ± 0.08
Palivizumab45.0474.32 ± 0.08
RSV-IGIV427.8643.49 ± 0.19
RSV-IGIV483.32952.0 ± 0
RSV-IGIV425014004.80 ± 0.08

Definitions: BSA = Bovine Serum Albumin; RSV-IGIV = RespiGam; SE = standard error.

A greater than 2 log reduction in RSV titer was obtained at a palivizumab dose of 1.67 mg/kg, corresponding to serum antibody levels of 11 and 13 mcg/mL (Experiments I and II respectively) at the time of RSV challenge when compared to the negative controls.

Additional experiments were performed to compare the potency of palivizumab against A and B subtypes of RSV in cotton rats dosed with palivizumab intramuscularly at doses of 0.625, 1.25, 2.5, or 5.0 mg/kg.

The results of all intramuscular studies show at least a 50-fold increase in potency with palivizumab when compared to RespiGam administered intramuscularly. Both A and B RSV subtypes were susceptible to palivizumab. A greater than 2 log reduction in RSV titer was obtained at doses of 1.67 to 2.5 mg/kg, corresponding to serum antibody levels of 11 to 21 mcg/mL at the time of RSV challenge.

Intravenous Administration for Prophylaxis in Cotton Rats

Three dose ranging studies evaluating palivizumab, administered by an intravenous route, for prophylaxis of RSV infection in cotton rats were performed. The purpose of these studies was to determine a serum concentration of palivizumab which results in at least a 99% (2 log10) reduction of RSV lung titer.

In the first two studies, cotton rats were anesthetised and infused intravenously with Bovine Serum Albumin (BSA) (10 mg/kg) or palivizumab at doses of 10 mg/kg, 5.0 mg/kg, 2.5 mg/kg, 1.25 mg/kg, 0.625 mg/kg, or 0.312 mg/kg. The third experiment was identical except that the 0.312 mg/kg dose of palivizumab was omitted. One day after infusion, the animals were anesthetised, bled and challenged by intranasal instillation with RSV. Serum palivizumab levels at the time of challenge were determined using ELISA. Four days after RSV challenge all animals were sacrificed, lungs were harvested, homogenized and RSV titers were determined. Table 8 represents the combined data from the three studies.

Table 8. Intravenous Prophylaxis in Cotton Rats (All Experiments Combined)"Determination of serum concentration of Palivizumab which results in at least a 99% (2 log10) reduction of RSV lung titer"
Compound Number of
Animals
Dose
(mg/kg)
Mean ± SE Concentration of Human
IgG at Challenge (mcg/mL)
Geometric Mean ± SE
Lung Viral Titer
(pfu/g)
BSA 18 10 0 1.3 x 105 ± 1.2
Palivizumab 7 0.312 2.67 ± 0.60 4.6 x 104 ± 1.5
Palivizumab 17 0.625 5.27 ± 0.27 2.7 x 104 ± 1.3
Palivizumab 18 1.25 10.1 ± 0.29 3.3 x 103 ± 1.4
Palivizumab 17 2.5 28.6 ± 2.15 9.6 x 102 ± 1.5
Palivizumab 15 5.0 55.6 ± 3.43 1.3 x 102 ± 1.2
Palivizumab 18 10.0 117.6 ± 5.09 1.0 x 102 ± 1.0

Definitions: BSA = Bovine Serum Albumin; SE = standard error.

The data indicated that a dose of 2.5 mg/kg and, more importantly, a corresponding circulating level of palivizumab of about 30 mcg/mL correlated with a greater than 2 log reduction in viral pulmonary RSV titer.

Potential Enhancement of Infection (cotton rats)

Potential Enhancement of Infection and/or Selection for Escape Mutants of RSV

The cotton rat model was used to determine whether the presence of palivizumab at non-inhibitory levels could enhance viral replication or virus-induced pathology during a primary RSV infection. In addition, the experiment was also designed to analyze the virus isolated from the lungs of the palivizumab-treated animals for the presence of antibody-resistant variants.

Animals each were administered BSA at 10 mg/kg (Group A) or palivizumab at 0.0032 mg/kg, 0.016 mg/kg, 0.08 mg/kg, 0.4 mg/kg, 2.0 mg/kg, and 10 mg/kg (Groups B through G), intramuscularly. Twenty-four hours later all animals were challenged with RSV. Four days later the animals were sacrificed and the lung tissue was analyzed for RSV levels, and prepared for histopathology. The results of this study are summarized in Table 9.

Table 9. Analysis of Potential Enhancement of Infection and/or Selection for Escape Mutants of RSV during Prophylaxis with Palivizumab in Cotton Rats
Group Dose
(mg/kg)
Mean Log ± SE RSV
Titer*
(pfu/g)
Animals with RSV related
pathology
Severity of lesions
observed
(N - 4)**
A 0 (BSA) 3.23 ± 0.08 4/4 1
B 0.0032 3.2 ± 0.02 4/4 1
C 0.016 3.3 ± 0.22 4/4 1
D 0.08 3.35 ± 0.16 3/4 0
E 0.4 2.98 ± 0.06 4/4 1
F 2.0 2.28 ± 0.16 2/4 0
G 10 <2 ± 0 0/4 0

* RSV Titer measured in 0.1 mg/mL BSA

** N (Normal), 1 (Minimal), 2 (Mild), 3 (Moderate), 4 (Marked).

Definitions: BSA = Bovine Serum Albumin; SE = standard error.

Lung changes consistent with RSV infection were found in four of four animals each from Groups A, B, C, and E, three of four animals from Group D, two of four animals from group F and none of four animals from group G. The data indicate that palivizumab protects from RSV-induced lesions in cotton rats at a dose of 2 mg/kg (partial protection) and 10 mg/kg (complete protection). No enhancement of pathology was observed at sub-effective doses of palivizumab. Based on these results, palivizumab does not induce antibody-dependent enhancement in a primary infection of cotton rats.

Re-challenge of Previously Infected Cotton Rats

Again using the cotton rat model, a study was conducted to analyze the potential for enhancement of pulmonary virus of RSV associated histopathology in previously infected animals. Cotton rats were divided into three Groups. Group 1 animals were administered 10 mg/kg BSA and Group 2 received 10 mg/kg palivizumab intravenously one day prior to challenge with RSV (105 pfu/Long strain). Group 3 was not dosed and was mock challenged with medium only. Four animals from each group were sacrificed four days after infection and pulmonary virus titers were determined. The remaining animals were bled bi-weekly until palivizumab was no longer detectable in Group 2 animals. At that time, the remaining animals were re-challenged with a low dose of RSV (103 pfu) and sacrificed on day four after challenge. Lungs were divided and prepared for histopathology and virus titration as previously described above. The results of the challenge and rechallenge are summarized in Table 10.

Table 10. Results of Re-challenge of Previously Infected Cotton Rats: Analysis for Potential Enhancement of Pulmonary Virus or RSV Associated Histopathology (Study No. III)
Group Dose 1° RSV challenge (mean
log pfu/g ± SE)
2° RSV challenge
(mean log pfu/g ± SE)
Animals with RSV
Associated
Pathology
Range of
Severity
(N - 4)
1 10 mg/kg
BSA
5.51 ± 0.05 < 2 ± 0 0/7 0
2 10 mg/kg
palivizumab
< 2 ± 0 < 2 ± 0 0/5 0-1
3 None Mock Challenged 2.46 ± 0.14 1/8 0-1

** N (Normal), 1 (Minimal), 2 (Mild), 3 (Moderate), 4 (Marked).

Definitions: BSA = Bovine Serum Albumin; SE = standard error.

Inflammation of the bronchiole consistent with RSV infection was diagnosed in one of eight untreated control animals but not any other animal of the study. Other lesions (inflammation associated with foreign material, histocytosis and pigmentation) were considered incidental lesions not related to treatment. Since the untreated controls (Group 3) had not received a primary challenge with RSV on study day one, the data suggest that the secondary challenge (rechallenge) with 103 pfu/animal of RSV did not induce extensive pulmonary lesions detectable microscopically four days after challenge. Viral titers were also consistent with a low level of infection in the Group 3 animals, as intended. Except for the foreign material inflammation of one animal of Group 2, lesions were not seen in the lungs of Group 1 and 2 animals indicating lack of residual changes attributable to RSV, BSA, or palivizumab. Thus, no enhancement of either virus replication or virus-induced pathology was observed in a secondary RSV infection. Furthermore, the animals protected by palivizumab from an initial RSV challenge were also shown to be resistant to a secondary challenge of RSV.

Pharmacokinetics

Non-clinical pharmacokinetics was assessed in the Cynomolgus monkey, a macaque surrogate of humans. These investigations revealed considerable interanimal variability but a consistent biphasic pattern, attributable to the distribution and the elimination phases. Some of this variability might be due in part to difficulties matching age, physical characteristics, health and nutritional status in these wild-caught monkeys. Although the elimination phase is quite long, the dosing strategy in humans specifies one month between each injection in order to assure acceptable trough levels.

Absorption

An intravenous infusion of liquid, unfiltered palivizumab at 10 mg/kg was provided to two female Cynomolgus monkeys (Figure 1). Standard hematology, clinical chemistry and urinalyses assays were performed at the initiation and conclusion of the study. Samples were removed from a contralateral vein for kinetic analysis at 5, 15, 30, 60, 120, 180, 360 and 720 minutes, as well as days 1, 2, 4, 7, 10, 15 and 21 post-dosing. The Cmax was approximately 200 mcg/mL or nearly 7 times the effective concentration in human plasma. Consistent with the study above, the distributional phase or alpha half-life was 8.8 to12.6 hours, while the elimination phase or beta half-life was approximately 8.6 days. There was no evidence for intolerance of this dosage in this model.

Kinetic data were also obtained during the acute toxicity study performed in Cynomolgus monkeys. Single dosages of liquid, unfiltered palivizumab at 10 and 30 mg/kg were given intravenously, equating up to two times the human maximum clinical dose, to two monkeys of each sex.



Figure 1. Pharmacokinetic Analysis of Palivizumab in Cynomolgus Monkeys

The pharmacokinetic curves in Figure 1 were generated from time course samples at 5, 15, 30, 60, 120, 180, 320, 720 minutes and day 1, 2, 4, 7, 15 and 20 for animal #1724 (open circles) and #1854 (solid triangles).

These dosages provided mean Cmax concentrations of 286 and 595 mcg/mL, respectively, approximately 10 and 20 times the effective concentration (30 mcg/mL) defined as efficacious in the cotton rat model and later confirmed in human trials. Considerable interanimal variability was noted in the biphasic half-life of the drug in these animals, with the alpha phase lasting from 0.24 hours to 30.66 hours; the beta phase was more prolonged, lasting from 4.2 days to 5.9 days. Thus, total clearance is quite protracted in the Cynomolgus monkey.

Human

Pharmacokinetics

Study MI-CP-097

The pharmacokinetics and safety of the SYNAGIS solution for injection and SYNAGIS lyophilized powder formulations, following 15 mg/kg intramuscular administration, were compared in a cross-over trial of 153 infants less than or equal to 6 months of age with a history of prematurity (less than or equal to 35 weeks gestational age). The ratio (solution:powder) of the trough serum concentrations was estimated to be 1.04 with a corresponding 90% confidence limit of (0.998 -1.083). The results of this trial indicated that the trough serum concentrations of palivizumab were similar between the solution for injection and the lyophilized powder formulations.

Extended Dose Study (Study W00-350)

An open, prospective safety and pharmacokinetics study examined the safety, tolerance and pharmacokinetics of SYNAGIS when administered for up to 7 months in Saudi Arabia, a subtropical region where the reported RSV season is frequently longer than in temperate countries. Eighteen preterm infants (< 34 weeks gestation), ranging in age from newborn to 29 weeks, with or without chronic lung disease (CLD), judged to be at risk for RSV infection, and palivizumab naïve, were included in the study. SYNAGIS 15 mg/kg was injected once per month, for up to 7 months during the RSV season. Safety data are based on all 18 subjects who received SYNAGIS, 17 of whom received all 7 doses.

Palizumab serum concentrations were not available for all subjects at all visits (Table 11). Target serum trough palivizumab levels (30 mcg/mL or greater) were achieved. No significant elevations of anti-palivizumab antibody titer were observed. These study results suggest that seven SYNAGIS doses are non-immunogenic and not associated with increased adverse events.

Table 11. Summary of SYNAGIS® Blood Assay Results (Study W00-350)
Study Visit* Number of subjects Mean ± Standard Deviation
≥ 30mcg/mL < 30mcg/mL < LOQ NRP Total
Visit 1 0 0 17 1 18 0 ± 0
Visit 2 16 0 1 1 18 44.72 ± 18.67
Visit 5 16 0 0 2 18 121.06 ± 36.23
Visit 7 14 0 0 4 18 144.36 ± 47.54

* Blood was drawn prior to study drug administration at each visit.

Definitions: LOQ = Limit of quantification; mcg/mL = mcg/mL of SYNAGIS; NRP = Not reported.

Microbiology

Laboratory Tests

Interference with immunologically-based RSV diagnostic assays by palivizumab has been observed in laboratory studies. Rapid chromatographic/enzyme immunoassays (CIA/EIA), immunofluorescence assays (IFA), and direct immunofluorescence assays (DFA) using monoclonal antibodies targeting RSV F protein may be inhibited. Therefore, caution should be used in interpreting negative immunological assay results when clinical observations are consistent with RSV infection. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay, which is not inhibited by palivizumab, may prove useful for laboratory confirmation of RSV infection. See (DRUG INTERACTIONS, Drug-Laboratory Interactions).

Antiviral Activity

The antiviral activity of palivizumab was assessed in a microneutralization assay in which increasing concentrations of antibody were incubated with RSV prior to addition of the human epithelial cells HEp-2. After incubation for 4 to 5 days, RSV antigen was measured in an enzyme-linked immunosorbent assay (ELISA). The neutralization titer (50% effective concentration [EC50]) is expressed as the antibody concentration required to reduce detection of RSV antigen by 50% compared with untreated virus-infected cells. Palivizumab exhibited median EC50 values of 0.65 mcg/mL (mean [standard deviation] = 0.75 [0.53] mcg/mL; n = 69, range 0.07 to 2.89 mcg/mL) and 0.28 mcg/mL (mean [standard deviation] = 0.35 [0.23] mcg/mL; n = 35, range 0.03 to 0.88 mcg/mL) against clinical RSV A and RSV B isolates, respectively. The majority of clinical RSV isolates tested (n = 96) were collected from subjects in the United States with the remainder from Japan (n = 1), Australia (n = 5) and Israel (n = 2). These isolates encoded the most common RSV F sequence polymorphisms found among clinical isolates worldwide.

Resistance

Palivizumab binds a highly conserved region on the extracellular domain of mature RSV F protein, referred to as antigenic site II or A antigenic site, which encompasses amino acids 262 to 275. All RSV mutants that exhibit resistance to palivizumab have been shown to contain amino acid changes in this region on the F protein. No known polymorphic or non-polymorphic sequence variations outside of the A antigenic site on RSV F protein have been demonstrated to render RSV resistant to neutralization by palivizumab. At least one of the palivizumab resistance-associated substitutions, N262D, K272E/Q, or S275F/L was identified in 8 of 126 clinical RSV isolates from subjects who failed immunoprophylaxis, resulting in a combined resistance-associated mutation frequency of 6.3%. A review of clinical findings revealed no association between A antigenic site sequence changes and RSV disease severity among children receiving palivizumab immunoprophylaxis who develop RSV lower respiratory tract disease. Analysis of 254 clinical RSV isolates collected from immunoprophylaxis-naïve subjects revealed palivizumab resistance-associated substitutions in 2 (1 with N262D and 1 with S275F), resulting in a resistance associated mutation frequency of 0.79%.

Toxicology

Acute Toxicity

Rabbits

New Zealand White rabbits were treated with intramuscular or subcutaneous injections of either 15 or 50 mg/kg of palivizumab or control vehicle (2x Formulation Buffer; 0.15 or 0.5 mL/kg) and sacrificed on Day 4 (interim) or Day 15 (terminal). Body weights were collected prior to dosing and prior to interim and terminal sacrifice, and weight changes were determined. Weight changes in the animals that were sacrificed on Day 4 or Day 15 were not adversely affected by any exposure route or any dose of either the vehicle control or palivizumab. Slight (Day 2) and very slight (Days 3 and 4) erythema was observed in one of eight animals treated with an intramuscular injection of 50 mg/kg of palivizumab (Group 5). Very slight erythema was also observed on Days 2 and 3 in one of the eight animals treated with a subcutaneous injection of 15 mg/kg of palivizumab (Group 4). A hematoma was observed on Day 2 in one of the eight animals treated with subcutaneous injection of 50 mg/kg of palivizumab (Group 6). The hematoma was not observed after Day 2. A lesion was observed during the necropsy (Day 4) in one of the four animals treated with an intramuscular injection of 0.15 mL/kg of the control vehicle (Group 1). Any lesions observed macroscopically are most likely attributable to trauma caused during the injection procedure. Microscopic evaluation of the injection site from animals necropsied on Days 4 and 15 confirmed that treatment with palivizumab did not result in any lesions attributable to the test article.

Tissue damage from palivizumab was evaluated in a Good Laboratory Practices-compliant study in New Zealand White rabbits. The lyophilized product was injected in vehicle both intramuscularly and subcutaneously into the thigh muscles of 2 rabbits/sex/group; one group served as a vehicle control (5.6% mannitol, 3.0 mM glycine, 47 mM histidine), while the other 2 received either 15 or 50 mg/kg injected in a bolus. The lower dosage is equivalent to the maximum recommended human dosage, while the higher dosage provided over three times the human dosage. The animals tolerated the treatments without evidence for systemic toxicity. On evaluation Days 4 and 15, some injection sites were erythematous, while most were unremarkable. One rabbit in the higher dosage group developed a hematoma at the injection site, presumably due to accidental injection into or near a major blood vessel. Histologic examination of the injection sites revealed no evidence for local intolerance.

Rats

A 14-day single dose toxicity study in Sprague-Dawley rats (6/sex/group) provided single, intravenous doses up to 840 mg/kg or 56 times the maximum human dose of 15 mg/kg. Based on pre-study body weights of male and female rats, dosages were adjusted to the mean weight of each group. Male rats received a 210 mg/kg (1.2 mL), 420 mg/kg (2.4 mL) or 840 mg/kg (5.0 mL) dose of palivizumab or a 5.0 mL injection of the buffer control solution. Female rats received a 210 mg/kg (1.0 mL), 420 mg/kg (1.9 mL) or 840 mg/kg (3.9 mL) dose of palivizumab or a 3.9 mL injection of the formulation buffer as a control. All doses of the test article were administered at a concentration of 57 mg/mL. Although the preferred route for clinical use is intramuscular, not intravenous, this parenteral route provided an efficacious response in cotton rats infected with RSV and higher maximum plasma concentrations (Cmax) achieved via the intravenous route were more likely to demonstrate systemic toxicity. These dosages were anticipated to provide up to 8 times the human exposure, based on surface area calculations. Cage side observations were recorded twice daily, and all rats were observed at approximately one hour after dose administration for mortality or pharmacotoxic signs, and weekly for clinical signs and abnormality. Ophthalmic examinations were performed during the pretreatment and prior to necropsy. These rats were observed for 14 days, including traditional assessments of clinical signs, body weight changes, food consumption, ophthalmoscopy, hematology, clinical and anatomic pathology. Despite these considerable multiples of the human exposure, there was no evidence for systemic toxicity. Superficial corneal lesions were described but attributed to the repeated bleeding via the infraorbital sinus.

Blood samples were collected and serum harvested for the sponsor during the pretreatment week, on Days 0, 1, 3, 5, 7 and prior to necropsy (Day 14). Blood samples were also collected for hematology evaluations during pretreatment, on Day 3 and at time of necropsy. Serum samples for clinical chemistry evaluations were collected during the pretreatment week (retained frozen for possible evaluation) and prior to necropsy. A complete necropsy was performed on all rats in all groups on Day 14. All retained tissues from animals in Groups 1 (0 mg/kg), 2 (210 mg/kg) and 4 (840 mg/kg) were processed and evaluated histologically. Since no treatment-related gross or histologic lesions were observed in these groups, tissues from animals in Group 3 (420 mg/kg) were not examined.

All animals survived until scheduled sacrifice. The only abnormal clinical signs noted were for one male rat animal in Group 1on Days 7 (exophthalmos and eye opacity) and 14 (eye opacity) and for one Group 1 female on Days 7 and 14 (eye opacity). These rats were judged to be clinically normal at all other physical examination intervals. No abnormal ophthalmic findings were noted before dosing. Panophthalmitis was observed in the right eye of one male rat and one female rat in Group 1. Retinal detachment was observed in the right eye of one Group 2 male and retinal detachment with hemorrhage was observed in the right eye of one Group 4 female. These lesions were considered secondary to previous blood collections from the infraorbital sinus. No significant changes in body weight or food consumption were observed during the study.

Statistically significant group differences, relative to control group values of the same sex, that were observed in clinical pathology data were considered to be chance occurrences and not indicative of a drug-related toxic effect. Findings noted at necropsy were not attributed to administration of the test material. No significant changes in group organ weights were observed. Histopathologically, no treatment-related lesions were observed in any tissue of any treatment group. Sporadic lesions commonly seen in these rats under laboratory conditions were rare.

Under the conditions of this study, a single intravenous injection of palivizumab, when administered to male or female rats at doses of 210, 420 or 840 mg/kg did not produce evidence of toxicity.

Cynomolgus Monkeys

The acute toxicity study of palivizumab administered intravenously to Cynomolgus monkeys consisted of three groups of two monkeys/sex/group. Group 1 animals were administered phosphate buffer saline (PBS) which served as control and Group 2 and 3 animals were dosed with 10 and 30 mg/kg of the test article, respectively (equating to 2 times the human maximum dose). Dose administration was performed via intravenous infusion through a percutaneous catheter placed in a peripheral vein of each monkey. Animals were individually restrained in slings and infusion of vehicle or test article was performed over a 15 minute period, without tranquillization, on Day 1 of the study.

Animals were observed for 14 or 29 days; one animal/sex/group was subjected to a complete gross necropsy on Days 15 and 30. Various parameters were analyzed to assess the toxicity of the test material.

Daily clinical observations revealed findings such as abrasions, scabs, erythema, bruises, swelling (on various body sites), pale mucous membranes, alopecia, salivation, and discoloured feces in test article-treated and vehicle-treated (which had the most findings) groups. These findings were not considered to be associated with treatment with the test article but appeared to be due to the multiple bleeding procedures and the associated stress and trauma. The body weight, food consumption, physical examination, blood pressure and body temperature measurements did not exhibit any remarkable changes that could be attributed to the test material.

Clinical pathology data analysis revealed decreasing hemoglobin (HGB) and hematocrit (HCT) values, especially in one Group 2 and two Group 1 and 3 females during the first few days after dosing. However, the animals were in the process of recovery from this deficiency by Day 8 as was demonstrated by the increased reticulocyte counts and the upward trend in HGB and HCT. These reductions were judged to have been due to repeated venoclysis for the pharmacokinetic analyses.

Analysis of serum chemistry parameters revealed high creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) values in all animals on Days 1 to 2 and in a few animals on Day 3 in both vehicle and test article treated groups. These elevations were thought to be due to the restraining procedure used for dose administration in these animals, which caused their muscle enzymatic activities to increase and were not considered to be related to treatment with the test article. A decrease in blood urea nitrogen (BUN) values in all Group 1 and 2 animals and Group 3 females on Day 3 only, was difficult to interpret but was not considered a test article effect since it was also found in concurrent control animals.

No remarkable treatment changes were found in blood coagulation and urinalysis data as compared to the control animals. Organ weight analysis did exhibit changes in several Group 2 and 3 tissue weights as compared to corresponding weights from Group 1 animals, but since the available data was only from one animal/sex/group, the significance of this variation could not be determined.

Gross necropsy observation showed many single red/purple foci around the saphenous vein of five monkeys on Day 15 and four monkeys on Day 30 and subcutaneous hemorrhage and edema in one Group 2 male. These findings were considered to be due to the catheterization procedure used for dose administration.

Microscopic examination revealed a golden brown globular pigment consistent with hemosiderin in the renal tubule and a few other organs of one Group 3 female. Similar pigment, but more consistent with lipofuscin, was found in the tubular epithelial cells of one Group 1 female. One Group 3 male and two females, one each in Groups 1 and 2, also exhibited renal tubular pigments on Day 30. These hemosiderin-like pigments were considered incidental, not related to palivizumab, and without consequence to the well-being of the primate. The above-mentioned Group 3 female also exhibited some crystalline material in its cortical tubules, the reason for which could not be specifically ascertained with the available data. Microscopic observation also revealed acute inflammation at the administration site in all animals and mild to moderate hemorrhage at the saphenous vein in the Group 1 and 2 males. Also observed were a trauma-related lesion and subcapsular focus in a section of liver in one Group 3 male and one Group 2 female, respectively.

There were no microscopic findings observed that could be attributed specifically to the test article.

The data obtained from this study did not exhibit any potential toxicity following an intravenous infusion of test material up to a dose level of 30 mg/kg in Cynomolgus monkeys when observed for 30 days.

Long-Term Toxicity

No long-term toxicity studies were performed, owing to the absence of tissue reactivity from palivizumab, the likely neutralization of the humanized antibody, the expectation of anaphylaxis or immune complex formation to the foreign protein, and the considerable time separation between human exposures.

Mutagenicity and Carcinogenicity

Carcinogenicity studies have not been performed with palivizumab.

Mutagenicity studies have not been performed with palivizumab, nor are they normally required for monoclonal antibody products.

Reproduction and Teratology

Reproduction studies have not been performed with palivizumab.