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PHARMACOKINETICS |
From the Department of Pharmacokinetics/Drug Metabolism (Dr Harrison) and the Department of Medical Operations (Dr Astry and former employees Drs Kumar and Yunis), 3M Pharmaceuticals, St Paul, Minnesota.
Address for correspondence: Lester I. Harrison, PhD, 3M Center Bldg 270-3S-05, St Paul, MN 55144; e-mail: liharrison{at}mmm.com.
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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dose response were observed for the 3 subcutaneous doses. Serum concentrations following the 2.0-mg oral dose were always lower than those following the same intravenous dose, and the oral route had a bioavailability of 26.5% ± 7.84%. Concentrations appeared to increase with oral dose; however, large variabilities in both the rate and extent of absorption were seen between individuals. Approximately 40% of an absorbed dose was excreted unchanged in the urine. Overall, the study suggests that subcutaneous administration may be an acceptable method to deliver 852A for systemic applications.
Key Words: Pharmacokinetics • 852A • imidazoquinoline • bioavailability • Toll-like receptor 7 agonist
), and enhanced antigen presentation.2 These immune-stimulating effects are hypothesized to mediate the antiviral and antitumor activities observed with these compounds, including effects on both innate and acquired immunity.2,3 One imidazoquinoline TLR7 agonist, imiquimod, is marketed as a 5% cream for the topical treatments of genital warts, actinic keratosis, and superficial basal cell carcinoma.4
852A is an imidazoquinoline that is more TLR7 specific than imiquimod in preclinical cellular models.5 852A is N-[4-(4-amino-2-ethyl-1H-imidazo [4,5c]quinolin-1-yl)butyl]methanesulfonamide (Figure 1). 852A directly stimulates cells in the innate immune system to produce immune regulatory and anticancer cytokines primarily associated with the TLR7 pathway. For example, peripheral blood mononuclear cells secrete IFN-, interleukin-1 receptor antagonist, and the chemokine CXCL10 (IP-10) in response to 852A.5,6
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The first clinical trial of 852A examined intravenous drug administration in patients with solid organ tumors refractory to standard therapy.6 In that trial, the threshold for pharmacological activity of 852A was 0.6 mg/m2 (eg, 
1 mg for a 1.7-m2 adult). Although intravenous administration is not uncommon for cancer treatments, oral and/or subcutaneous administration may allow greater convenience for administration by health care staff and/or by patients.
This publication includes the results of a phase I clinical study that focused on the systemic delivery of 852A to healthy adult volunteers. The pharmacokinetics and bioavailability of this molecule were assessed following subcutaneous and oral administrations and compared with intravenous administration. Pharmacodynamics was assessed by measurements of IFN- and C-reactive protein (CRP).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The study consisted of 3 cohorts, each containing 6 subjects. A subject participated in only 1 cohort and received up to 3 doses of 852A as follows:
Subjects were domiciled the night prior to each dose visit within a hospital setting until the 24-hour postdose procedures were completed. A minimum 5-day washout period was maintained between each dose visit.
852A was supplied as a 0.2% (2 mg/mL) sterile solution for injection (3M Pharmaceuticals, St Paul, Minnesota). For intravenous dosing, drug solution was administered as a bolus over 10 seconds. For subcutaneous dosing, drug was administered into the thigh; injection sites were rotated. For oral dosing, drug was prepared in 50 mL of 5% dextrose for injection solution; following an overnight fast, the subject drank the prepared solution, followed by 150 mL of water. Food was allowed 4 hours later.
Safety assessments during this study included physical examinations, vital sign measurements, 12-lead electrocardiograms, routine laboratory tests including complete blood count with differential and platelet count, serum chemistry panel and urinalysis, and monitoring of adverse events and con-comitant medication use.
Blood samples were collected predose and following each dosing route for 24 hours: at 5, 15, and 30 minutes and 1, 2, 4, 6, 9, 12, 18, and 24 hours following intravenous administration; at 15, 30, and 45 minutes and 1, 2, 4, 6, 9, 12, 18, and 24 hours following subcutaneous administration; at 30 minutes and 1, 1.5, 2, 4, 6, 9, 12, 18, and 24 hours following oral administration. Prepared sera were analyzed for 852A content and, in some cases, for IFN- and CRP. Complete urine collections for the intervals of 0 to 12 hours and 12 to 24 hours were done following all doses.
Bioanalytical Procedures
The bioanalytical procedures for 852A were developed by and performed at 3M Pharmaceuticals. 852A was extracted from a 0.100-mL sample of serum or urine and quantified using reversed-phase isocratic liquid chromatography coupled to a tandem mass spectrometry system operated with turbo-ion spray in the positive-ion mode. Another proprietary imidazoquinoline, with similar mass and structure (variations in sidechains) and an elution profile that did not interfere with 852A, was included as an internal standard in the solid-phase extraction step. Liquid chromatography was performed with a 4.6 x 150-mm Zorbax Eclipse XDB C8 column (Agilent Technologies, Santa Clara, California), 5-µm particle size. For the serum analyses, the mobile phase was 0.1% formic acid in methanol/20 mM ammonium formate (pH 3; 55:45, v/v), and the flow rate was 1 mL/min. For urine, the mobile phase solvents of 0.1% formic acid in methanol and Milli-Q water (Millipore, Billerica, Massachusetts) were combined using a gradient program. The 852A transition from m/z 362 to m/z 213 was monitored for both matrices. Quality control standards at low, mid, and high concentrations were run at the beginning and end of all analytical runs.
The bioanalytical method was validated with respect to linearity, specificity, intraday and interday precision, accuracy, and recovery, according to the Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation.8 The interday precision and accuracy were calculated from all the analyses of the quality control standards that were analyzed during the study. The coefficient of variability for each concentration standard varied between 10% and 12% for both serum and urine; the relative error varied between -1.3% and 3.6% for both serum and urine. Freeze-thaw stability was demonstrated under the same conditions as the unknown samples. The lower limits of quantitation were 0.100 ng/mL in serum and 0.200 ng/mL in urine.
IFN- was analyzed by MDS Pharma Services (Montreal, Canada), using a commercially available ligand-binding assay (PBL Biomedical Laboratories, Piscataway, New Jersey). This antibody-based assay was designed to detect 13 of 14 known subtypes of IFN- and was validated with respect to accuracy, precision, and stability. The lower limit of quantitation was 3.15 IU/mL. Results were supported by independent analyses with another IFN- bioanalysis method at 3M. Serum CRP levels were measured by the University of Minnesota Outreach Laboratories (Minneapolis), using an immunoturbinometric assay kit (Roche Diagnostics, Basel, Switzerland). The lower sensitivity limit for the assay was 0.30 mg/dL.
Data Analyses
Pharmacokinetic parameters were estimated by non-compartmental analysis using a validated software program (Kinetica 3.0, Thermo Fisher Scientific, Waltham, Massachusetts). The primary pharmacokinetic parameters were the maximum serum concentration (Cmax) or, in the case of intravenous administration, the serum concentration at 5 minutes (C5 min), as well as the area under the serum concentration-time curve from time 0 to the time of last quantifiable concentration (AUC0-t) and from time 0 to infinity (AUC0-
). Other pharmacokinetic parameters calculated included the time of Cmax (tmax), the apparent elimination half-life (t
), volume of distribution (Vd), and total body clearance (CL). For the intravenous dose, the concentration at time 0 (C0) was also calculated by extrapolation.
Based on internal standard operating procedures, a calculated t was reportable if at least 3 measurable serum concentrations were available after Cmax and the correlation coefficient of the regression line was greater than 0.922. Similarly, AUC0- was reportable if the extrapolated area was less than 20% of the total.
Absolute bioavailability was assessed in cohort 2 by comparing AUC0- calculated following the subcutaneous and oral doses to that following the reference intravenous dose in the same individual. The 95% confidence interval (CI) for each AUC comparison was also calculated. For the proportionality analyses, the hypothesis that the slope of the fitted least squares linear regression line supported a 1:2:4 ratio for the subcutaneous doses or a 1:1.5:2 ratio for the oral doses was tested with a power model. Proportionality was assumed if the hypothesis could not be rejected.
For the IFN- and CRP measurements, measured concentrations were not considered reportable unless they were at least twice the lower limit of quantitation of the respective analyte.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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of approximately 7 to 9 hours and a Vd of 5 to 6 L/kg.
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Proportionality of Subcutaneous Doses
Serum concentration of 852A increased linearly with each of 3 rising subcutaneous doses administered in cohort 1 (Figure 3). The range of tmax values varied between 0.25 hours (time of the first postdose sample) and 0.75 hours in these subjects. Proportional increases in the derived pharmacokinetic parameters with subcutaneous dose, with correlation coefficients for the least squares lines of pharmacokinetic parameter versus dose of approximately 0.92, were seen (Table II).
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Proportionality of Oral Doses
Based on an interim assessment of the bioavailability and safety of the 2.0-mg oral dose from cohort 2, a starting oral dose of 10.0 mg was selected for cohort 3. As this dose was well tolerated, the second dose of cohort 3 was increased to 20.0 mg. An adverse event attributed to study drug occurred several days postdosing in 1 subject at this dose level; all scheduled blood collections were obtained for this subject and therefore included in analyses of the 20.0-mg dose. The third cohort dose was de-escalated from the planned 30.0-mg dose to a 15.0-mg dose.
Overall, serum concentrations of 852A appeared to increase with the dose following oral administrations of an 852A solution (Figure 4). However, large variabilities in both the rate and extent of absorption were seen between individuals. As a result, there were no statistical differences found between doses for any of the pharmacokinetic parameters following oral administration (Table III). The unusual profile following the 15-mg dose shown in Figure 4 was the result of 2 subjects having initial concentrations 2- to 5-fold greater than the other subjects. No AUC0- data were reported for this cohort because in most subjects at all doses, the extrapolated areas were greater than 20% of the total.
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The subject with the adverse event had the highest serum Cmax and AUC values of the 6 subjects receiving the 20.0-mg dose. Although the Cmax in this individual was high (18.1 ng/mL), it was still 2- to 4-fold less than the Cmax values observed to be well tolerated in all subjects in this study following the intravenous dose and comparable to the Cmax values found to be well tolerated in the other subjects given oral doses. The AUC0-t value (297 ng/mL/h), on the other hand, was more than double that observed in any other subject in the study.
Urinary Excretion
Table IV summarizes the percentage of 852A excreted in the urine as unchanged drug for the various doses administered by the 3 routes studied. The intravenous results show that 40% of the drug in the body is excreted unchanged in the urine. The lower excretion amounts for the other routes reflect their bioavailabilities. The amount of 852A excreted by the withdrawn subject in cohort 3 was comparable to others in the same dose group.
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The percentage of urinary excretion mean ± SD reported in Table IV following the 2.0-mg subcutaneous dose represents the contributions from cohort 1 (34.7% ± 2.18%) and from cohort 2 (44.5% ± 12.6%). The reason for the recovery difference between the cohorts is unknown. However, the observed recovery for this dose in the subjects of cohort 1 (n = 3) was consistent with the recoveries for the other 2 subcutaneous doses administered to this cohort; likewise, the observed recovery for the 2.0-mg subcutaneous dose in the subjects of cohort 2 (n = 4) was comparable to the recovery following the same intravenous dose administered to cohort 2. These observations suggest that differences in urinary recoveries between cohorts 1 and 2 might be due to the subject populations.
Pharmacodynamic Measures
Serum concentrations of IFN- were evaluated as the primary indicator of 852A pharmacologic activity in cohorts 1 and 2. IFN- responders were defined as subjects having at least 1 IFN- concentration after 852A administration that was greater than twice the lower limit of quantitation (>6.3 IU/mL). For the intravenous dose, 3 of 6 subjects (50%) were IFN- responders. Following the subcutaneous administrations, 0 of 6 subjects (0%) were classified as IFN- responders following the 0.50-mg dose, 1 of 6 subjects (17%) responded following the 1.0-mg dose, and 4 of 9 subjects (44%) responded following the 2.0-mg dose. Following the oral dose of 2.0 mg, 0 of 4 subjects (0%) were classified as IFN- responders.
Serum levels of CRP, another indicator of cytokine induction, were measured in cohorts 2 and 3 at predose and 24 hours postdose. Elevated CRP concentrations at least twice the lower limit of quantitation (>0.60 mg/dL) were observed in 2 of 6 (33%) subjects given the intravenous dose and in 0 of 5 (0%) subjects given the 2.0-mg subcutaneous dose. Following oral administration, elevated CRP concentrations were observed in 0 of 5 (0%), 0 of 6 (0%), 1 of 5 (20%), and 2 of 6 (33%) subjects when given 2.0, 10.0, 15.0, and 20.0 mg, respectively. The 24-hour CRP concentration of the subject in cohort 3 who discontinued treatment was >4-fold than the measurable concentration in any other subject.
Safety
For cohort 1, 33% (2/6) of subjects experienced at least 1 adverse event possibly or probably related to study drug after the 0.5-mg dose, 83% (5/6) after the 1.0-mg dose, and 100% (3/3) after the 2.0-mg dose. The most commonly reported adverse events by system organ class were administration site conditions (injection site pain) and general disorders (chills, pain). For cohort 2, 67% (4/6) of subjects experienced at least 1 adverse event possibly or probably related to study drug after intravenous dosing, 50% (3/6) after subcutaneous dosing, and 40% (2/5) after oral dosing. The most commonly reported adverse events by system organ class were nervous system disorders (dizziness, headache). For cohort 3, 33% (2/6) of subjects experienced at least 1 adverse event possibly or probably related to study drug after the 10.0-mg dose, 20% (1/5) after the 15.0-mg dose, and 50% (3/6) after the 20.0-mg dose. The most commonly reported adverse events were nervous system disorders (headache). No severe adverse events were reported in the study.
Transient, dose-dependent increases in white blood cell and absolute neutrophil counts and a decrease in mean absolute lymphocyte count were observed following all routes of administration. These cellular count changes peaked by 6 to 12 hours and trended toward reversal to baseline by 24 hours. No other safety measure was remarkable.
One subject enrolled in cohort 3, a 41-year-old woman, experienced a mild headache following the 10.0-mg oral dose and a moderate headache, as well as fever, chills, body aches, anxiety, nausea, vomiting, pallor, palpitations, and dizziness, following the 20.0-mg dose. These adverse events were considered to be possibly or probably related to study drug. This subject was withdrawn from the study 3 days later after receiving her last dose due to hypotension (blood pressure of 86/48 mm Hg) and tachycardia (pulse rate of 142 bpm); these were resolved at the visit 1 week after the dose.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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could not be determined in cohort 3. In addition, because of subject discontinuations or incomplete sample collections, some pharmacokinetic parameters were estimated based on only 3 values. However, the study was successful in providing the needed information to select subcutaneous administration as the preferred route for continued evaluation.
Subcutaneous administration appeared to be a well-tolerated and efficient route to administer 852A for systemic applications. The most common adverse event was injection site pain. This route had an 80% absolute bioavailability and gave serum concentrations comparable to intravenous administration by 30 minutes. Predictable, linear kinetics and an IFN- dose response were also observed.
The expectation based on animal data that 852A metabolism would not be extensive in subjects is supported by the observation that 40% of an intravenous 852A dose was excreted as unchanged drug in the urine. A mass balance study would be needed to confirm this. It is possible that limited metabolism of 852A (if confirmed) could explain the finding that its elimination half-life was approximately 4 times that reported for imiquimod, a drug with extensive metabolism.7
Administration of 852A as an oral solution was intended to maximize oral absorption by omitting the added dissolution process of a solid dose form. Even from solution, 852A absorption was low and variable, within a subject as well as between subjects. The cause of this variability is likely due to the vagaries in the gastrointestinal absorption process of a compound that has a pKa around that of physiological pH (7.3), rather than first-pass metabolism, as the biotransformation of 852A does not appear to be extensive. Similar variability following oral administration has been reported for imiquimod.7 One subject withdrew because of an adverse event following the 20.0-mg oral dose; however, given the small number of subjects evaluated at this dose, further study in additional subjects may be required before drawing conclusions on oral tolerability.
The observation that systemic 852A administration was associated with transient and reversible decreases in mean lymphocyte counts has been seen with other imidazoquinolines. Generalized induction of vascular adhesiveness, resulting in trapping of peripheral leukocytes either at the blood vessel wall or within peripheral tissues, has been observed with resiquimod in a murine model.9
The serum drug levels associated with the withdrawal of a subject following the 20-mg oral dose were below those expected to result in significant cytokine induction. However, the gut is a rich source of immune cells that can be directed to produce a variety of cytokines, and TLR7 has been reported to present on hepatocytes.10-13 It is possible, therefore, that oral administration of 852A stimulated sufficient local cytokine induction from responsive cells in the intestine or liver to "spill over" into the systemic circulation, resulting in the observed adverse effects. A similar spillover phenomenon has been reported following topical administration of imiquimod.14 Although the elevation in CRP observed in the subject was suggestive of cytokine induction, IFN- was not measured in cohort 3 subjects.
Overall, the observations of measurable serum IFN- and changes in circulating white cell concentrations following intravenous and subcutaneous administrations are consistent with 852A functioning as a TLR7 agonist in humans.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Financial disclosure: This study was sponsored by 3M Pharmaceuticals and conducted at MDS Pharma Services. Drs Harrison, Astry, Kumar, and Yunis were employees of 3M Pharmaceuticals when the study was conducted.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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1. Stanley MA. Imiquimod and the imidazoquinolines: mechanism of action and therapeutic potential. Clin Exper Dermatol. 2002;27: 571-577.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
2. Schiller M, Metze D, Luger TA, Grabbe S, Gunzer M. Immune response modifiers—mode of action. Exp Dermatol. 2006;15: 331-341.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
3. Schon M, Schon MP. The antitumoral mode of action of imiquimod and other imidazoquinolines. Curr Med Chem. 2007; 14: 681-687.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
4. Gupta AK, Cherman AM, Tyring SK. Viral and nonviral uses of imiquimod: a review. J Cutan Med Surg. 2004;8: 338-352.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
5. Gorden KB, Gorski KS, Gibson SJ, et al. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol. 2005;174: 1259-1268.
6. Dudek A, Yunis C, Kumar S, Harrison L, Hawkinson R, Miller J. Immune response activation with toll-like receptor agonist: results of a phase 1 study. J Clin Oncol. 2005;23(Suppl 16S): 2515.
7. Soria I, Myhre P, Horton V, et al. Effect of food on the pharmacokinetics and bioavailability of oral imiquimod relative to a subcutaneous dose. Int J Clin Pharmacol Ther. 2000;38: 476-481.[Web of Science][Medline] [Order article via Infotrieve]
8. Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. Rockville, Md: Food and Drug Administration; May 2001.
9. Gunzer M, Riemann H, Basoglu Y, et al. Systemic administration of a TLR7 ligand leads to transient immune incompetence due to peripheral-blood leukocyte depletion. Blood. 2005;106: 2424-2432.
10. Monteleone G, Vavassori P, Biancone L, Monteleone G, Pallone F. Immunoregulation in the gut: success and failures in human disease. Gut. 2002;50(Suppl 3): iii60-iii64.
11. Lee J, Wu CC, Lee KJ, et al. Activation of anti-hepatitis C virus responses via Toll-like receptor 7. Proc Natl Acad Sci USA. 2006;103: 1828-1833.
12. Schwabe RF, Seki E, Brenner DA. Toll-like receptor signaling in the liver. Gastroenterology. 2006;130: 1886-1900.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
13. Yrlid U, Cerovic V, Milling S, Jenkins CD, Klavinskis LS, MacPherson GG. A distinct subset of intestinal dendritic cells responds selectively to oral TLR 7/8 stimulation. Eur J Immunol. 2006;36: 2639-2648.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
14. Harrison LI, Skinner SL, Marbury TC, et al. Pharmacokinetics and safety of imiquimod 5% cream in the treatment of actinic keratoses of the face, scalp, or hands and arms. Arch Dermatol Res. 2004;296: 6-11.[Web of Science][Medline]
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