JOURNAL OF INTERFERON RESEARCH 5:423-428 (1985) Mary Ann Liebert, Inc., Publishers
A Comparison of Interferon Responses to Poly ICLC in Males and Females CHRISTOPHER T. BEVER, JR.,1 DALE E. McFARLIN,1 and HILTON B. LEVY2
ABSTRACT Interferon (IFN) responses to polyriboinosinic acid polyribocytidylic acid in poly-L-lysine and carboxymethylcellulose (poly ICLC) have been studied in detail in 6 men and 3 women as part of a preliminary trial in patients with multiple sclerosis (MS). Patients received intravenous (i.v.) doses of 100 /¿g/kg poly ICLC, and serum IFN levels were determined serially every 4 h for 16 h. Men and women produced substantial levels of IFN at 8, 12, and 16 h after infusion, but levels in men were consistently and significantly higher (p < 0.05). Interferon responses were examined also in 3 male and 3 female Rhesus monkeys. Serum samples were obtained 8 and 24 h after i.v. injections of 1 mg/kg of poly ICLC. Again, there were significantly higher levels of IFN in males. The observed differences may reflect sex-linked differences in either drug metabolism or specific sensitivity to IFN induction by poly ICLC. The most interesting possibility is that the difference is due to a more general difference in IFN response between males and females. Studies are currently in progress to evaluate these possibilities.
INTRODUCTION
polyribocytidylic acid is Polyriboinosinic (IFN)-inducing agent
in poly-L-lysine and carboxymethylcellulose (poly ICLC) interferon that has recently been cleared for investigational use in humans. The parent compound polyriboinosinic polyribocytidylic acid has well-known IFN-inducing abilities in vivo in nonprimates and in human cells in v/iro,'1' but early clinical trials demonstrated that little IFN was induced in primates,'2' because of rapid enzymatic hydrolysis by serum nucleases.'3' The addition of poly-L-lysine and carboxymethyl cellulose resulted in a derivative that resisted enzymatic hydrolysis'4' and had potent IFN-inducing abilities in primates'4' and humans.'5' The drug has now been used extensively in humans in phase-I and -II cancer trials.'6' For the past 2 years, IFN responses have been studied in patients receiving poly ICLC in a preliminary trial of the drug in multiple sclerosis (MS). " 8> A significant difference has been found between the IFN responses of males and females. an
'Neuroimmunology Branch, NINCDS, NIH, Bethesda,
Frederick, MD.
Preliminary results presented California, April 25, 1985.
at the
MD and
laboratory of Viral Diseases, NIAID,
Federation of American Societies for Experimental Biology, Anaheim,
85
BEVERETAL.
METHODS Patient Selection: Six men and four women, ages 23-54, with clinically definite MS'9' were selected as part of a preliminary trial of poly ICLC. "8' All patients were free of complicating medical illnesses and received no medications, other than those outlined below. All patients considered in the present study were hospitalized at the Clinical Center of the National Institutes of Health and informed consent was obtained for participation.
Drug Administration to Patients: Polyriboinosinic acid polyribocyticylic acid in polylysine and carboxymethylcellulose was obtained in 10-mg vials. Immediately before use, an aliquot was measured out to give the desired dose and was diluted in 50 cc of normal saline. The resulting suspension was administered by i.v. infusion over 30 min. Serum samples were then obtained at 4-h intervals for up to 24 h'. All patients received acetaminophen at the time of the drug infusion and every 4 h thereafter. Patients also received perchlorproperazine at the time of drug infusion and at 6 and 12 h. Patients were treated weekly starting with a 20-/¿g/kg dose and the dose was increased until they reached 100 ttg/kg. Thereafter, doses were given at weekly, biweekly, or monthly intervals. Only IFN data from 100 /¿g/kg doses at poly ICLC were included in the analysis. Data from analysis of IFN levels showed that the differences in interdose interval did not affect the observed levels so all data were included in the statistical analysis. One of the four women treated was excluded from the analysis because she was never able to tolerate the full 100 /xg/kg dose.
Study of Rhesus Monkey IFN Responses: Six-month-old Rhesus monkeys (3 male and 3 female) maintained in accordance with the Guide for the Care and Use of Laboratory Animals (DHEW Pub. #NIH 78-23). Each animal received an i.v. infusion of 1 mg/kg of poly ICLC in saline over 3 min while under Ketamine anethesia. Serum samples were obtained at 8 and 24 h. No other drugs were given. were
IFN Assay: Serum
samples were frozen and run in groups using as reference IFN the NIAID re#G023901537. Determinations were done by the method of Oie'10' by the CPE reducagent catalog tion method on human foreskin fibroblast cells infected with vesicular stomatitis virus. Data Analysis: In the MS patients, interferon levels at 4, 8, 12, and 16 h after drug infusion were available from multiple infusions. These values were used to calculate a mean log IFN at each time point for each patient. These means were then ranked and men and women compared using the Wilcoxin rank order test. For display purposes, a grand mean and standard deviation was computed for men and women at each time point. The IFN levels in the 3 male and 3 female Rhesus monkeys at each time point were compared using Student's t test.
RESULTS Interferon responses were greater in male than in female patients. Mean log IFN levels induced by poly ICLC 100 /¿g/kg for each patient and grand means and standard deviations for men and women are shown in the Fig. 1. The Wilcoxin rank order test showed that men produced significantly higher IFN levels at 8 and 12 h (p < 0.05). Because of concern that differences in IFN responses between the two groups might be related to some factor other than sex, the parameters of age, disease duration, severity of disability, disease activity, and prior treatment were examined (Table 1). No significant differences were found. Plots of IFN response versus age and IFN response versus disease duration (not shown) suggested no relationship and linear regression analysis confirmed that impression. Because treatment intervals of from 1 to 4 weeks were used and refractoriness of IFN induction has been demonstrated with repeated stimulation, the relationship of IFN level to intertreatment interval
86
SEX-LINKED DIFFERENCES IN INTERFERON RESPONSE
2.8
r
2.4 U Men 2.0
h
1.6
h Women
o
z
A Comparison of Interferon Responses to Poly ICLC in Males and Females CHRISTOPHER T. BEVER, JR.,1 DALE E. McFARLIN,1 and HILTON B. LEVY2
ABSTRACT Interferon (IFN) responses to polyriboinosinic acid polyribocytidylic acid in poly-L-lysine and carboxymethylcellulose (poly ICLC) have been studied in detail in 6 men and 3 women as part of a preliminary trial in patients with multiple sclerosis (MS). Patients received intravenous (i.v.) doses of 100 /¿g/kg poly ICLC, and serum IFN levels were determined serially every 4 h for 16 h. Men and women produced substantial levels of IFN at 8, 12, and 16 h after infusion, but levels in men were consistently and significantly higher (p < 0.05). Interferon responses were examined also in 3 male and 3 female Rhesus monkeys. Serum samples were obtained 8 and 24 h after i.v. injections of 1 mg/kg of poly ICLC. Again, there were significantly higher levels of IFN in males. The observed differences may reflect sex-linked differences in either drug metabolism or specific sensitivity to IFN induction by poly ICLC. The most interesting possibility is that the difference is due to a more general difference in IFN response between males and females. Studies are currently in progress to evaluate these possibilities.
INTRODUCTION
polyribocytidylic acid is Polyriboinosinic (IFN)-inducing agent
in poly-L-lysine and carboxymethylcellulose (poly ICLC) interferon that has recently been cleared for investigational use in humans. The parent compound polyriboinosinic polyribocytidylic acid has well-known IFN-inducing abilities in vivo in nonprimates and in human cells in v/iro,'1' but early clinical trials demonstrated that little IFN was induced in primates,'2' because of rapid enzymatic hydrolysis by serum nucleases.'3' The addition of poly-L-lysine and carboxymethyl cellulose resulted in a derivative that resisted enzymatic hydrolysis'4' and had potent IFN-inducing abilities in primates'4' and humans.'5' The drug has now been used extensively in humans in phase-I and -II cancer trials.'6' For the past 2 years, IFN responses have been studied in patients receiving poly ICLC in a preliminary trial of the drug in multiple sclerosis (MS). " 8> A significant difference has been found between the IFN responses of males and females. an
'Neuroimmunology Branch, NINCDS, NIH, Bethesda,
Frederick, MD.
Preliminary results presented California, April 25, 1985.
at the
MD and
laboratory of Viral Diseases, NIAID,
Federation of American Societies for Experimental Biology, Anaheim,
85
BEVERETAL.
METHODS Patient Selection: Six men and four women, ages 23-54, with clinically definite MS'9' were selected as part of a preliminary trial of poly ICLC. "8' All patients were free of complicating medical illnesses and received no medications, other than those outlined below. All patients considered in the present study were hospitalized at the Clinical Center of the National Institutes of Health and informed consent was obtained for participation.
Drug Administration to Patients: Polyriboinosinic acid polyribocyticylic acid in polylysine and carboxymethylcellulose was obtained in 10-mg vials. Immediately before use, an aliquot was measured out to give the desired dose and was diluted in 50 cc of normal saline. The resulting suspension was administered by i.v. infusion over 30 min. Serum samples were then obtained at 4-h intervals for up to 24 h'. All patients received acetaminophen at the time of the drug infusion and every 4 h thereafter. Patients also received perchlorproperazine at the time of drug infusion and at 6 and 12 h. Patients were treated weekly starting with a 20-/¿g/kg dose and the dose was increased until they reached 100 ttg/kg. Thereafter, doses were given at weekly, biweekly, or monthly intervals. Only IFN data from 100 /¿g/kg doses at poly ICLC were included in the analysis. Data from analysis of IFN levels showed that the differences in interdose interval did not affect the observed levels so all data were included in the statistical analysis. One of the four women treated was excluded from the analysis because she was never able to tolerate the full 100 /xg/kg dose.
Study of Rhesus Monkey IFN Responses: Six-month-old Rhesus monkeys (3 male and 3 female) maintained in accordance with the Guide for the Care and Use of Laboratory Animals (DHEW Pub. #NIH 78-23). Each animal received an i.v. infusion of 1 mg/kg of poly ICLC in saline over 3 min while under Ketamine anethesia. Serum samples were obtained at 8 and 24 h. No other drugs were given. were
IFN Assay: Serum
samples were frozen and run in groups using as reference IFN the NIAID re#G023901537. Determinations were done by the method of Oie'10' by the CPE reducagent catalog tion method on human foreskin fibroblast cells infected with vesicular stomatitis virus. Data Analysis: In the MS patients, interferon levels at 4, 8, 12, and 16 h after drug infusion were available from multiple infusions. These values were used to calculate a mean log IFN at each time point for each patient. These means were then ranked and men and women compared using the Wilcoxin rank order test. For display purposes, a grand mean and standard deviation was computed for men and women at each time point. The IFN levels in the 3 male and 3 female Rhesus monkeys at each time point were compared using Student's t test.
RESULTS Interferon responses were greater in male than in female patients. Mean log IFN levels induced by poly ICLC 100 /¿g/kg for each patient and grand means and standard deviations for men and women are shown in the Fig. 1. The Wilcoxin rank order test showed that men produced significantly higher IFN levels at 8 and 12 h (p < 0.05). Because of concern that differences in IFN responses between the two groups might be related to some factor other than sex, the parameters of age, disease duration, severity of disability, disease activity, and prior treatment were examined (Table 1). No significant differences were found. Plots of IFN response versus age and IFN response versus disease duration (not shown) suggested no relationship and linear regression analysis confirmed that impression. Because treatment intervals of from 1 to 4 weeks were used and refractoriness of IFN induction has been demonstrated with repeated stimulation, the relationship of IFN level to intertreatment interval
86
SEX-LINKED DIFFERENCES IN INTERFERON RESPONSE
2.8
r
2.4 U Men 2.0
h
1.6
h Women
o
z
