A proof-of-concept and drug-drug interaction study of pamapimod, a novel p38 MAP kinase inhibitor, with methotrexate in patients with rheumatoid arthritis
Abstract
This study was meticulously designed to thoroughly evaluate the potential for pharmacokinetic interactions between pamapimod, a targeted inhibitor of p38 mitogen-activated protein kinase, and methotrexate (MTX) when these two therapeutic agents are administered concurrently to patients suffering from rheumatoid arthritis (RA). Beyond the assessment of drug-drug interactions, a secondary but equally crucial objective of this investigation was to ascertain the preliminary pharmacodynamic effects of pamapimod, providing initial insights into its biological activity in a clinical setting. Given that methotrexate serves as a cornerstone disease-modifying antirheumatic drug (DMARD) in the long-term management of rheumatoid arthritis, understanding any potential alterations in its absorption, distribution, metabolism, or excretion when combined with a novel agent like pamapimod is paramount for ensuring both patient safety and the continued efficacy of their established treatment regimen.
The methodological framework for this clinical trial involved the enrollment of twenty-two patients diagnosed with rheumatoid arthritis who were already stabilized on a consistent and ongoing regimen of methotrexate, administered weekly at doses ranging from 10 to 25 milligrams, typically on specific days such as day 1 and day 8 of a cycle. These patients were then randomized into two distinct treatment arms: a larger cohort of seventeen patients received 300 milligrams of pamapimod once daily, while a smaller control group of five patients received a placebo once daily. The administration of either pamapimod or placebo commenced on day 5 and continued for a duration of ten consecutive days, concluding on day 14 of the study period. To enable a comprehensive pharmacokinetic assessment, biological samples, specifically blood and urine, were systematically collected at predetermined time points both before and after drug administration on key study days. These critical sampling days included day 1, when only methotrexate was administered, serving as a baseline for MTX pharmacokinetics; day 7, when pamapimod was administered as a monotherapy after the initial several days of dosing, allowing for its individual pharmacokinetic characterization; and crucially, day 8, when both methotrexate and pamapimod were coadministered, enabling the direct evaluation of any potential drug-drug interactions.
The results of this rigorous investigation provided reassuring findings regarding the pharmacokinetic compatibility of the two drugs. Notably, no clinically significant changes were observed in the plasma exposures, indicative of the overall drug levels in the bloodstream, nor in the renal clearance rates for either pamapimod, methotrexate, or their respective metabolites. This consistent pharmacokinetic profile held true regardless of whether the drugs were administered separately as monotherapies or concomitantly as a combination, implying that pamapimod does not appreciably alter the absorption, distribution, metabolism, or excretion of methotrexate, and vice-versa. From a safety perspective, the combination of pamapimod at a dose of 300 milligrams once daily for ten days, administered alongside weekly methotrexate, was generally well tolerated by the patient cohort. This indicates a favorable safety profile for this combination within the duration of the study, without unexpected or severe adverse events. Furthermore, an encouraging trend was observed in the pharmacodynamic effects, which reflect the drug’s impact on disease activity. Parameters commonly used to assess rheumatoid arthritis disease progression, including the tender joint count, swollen joint count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels, all generally exhibited a decrease between day 5, when pamapimod administration began, and day 14, the conclusion of the treatment period. This suggests a beneficial trend towards reduced inflammatory activity and improved clinical signs of the disease.
In conclusion, the findings from this pivotal study provide strong evidence that dose adjustments for either pamapimod or methotrexate are not necessary when these two medications are administered concurrently to patients with rheumatoid arthritis, significantly simplifying clinical management. Moreover, the observed general decrease in key pharmacodynamic markers of disease activity offers preliminary validation of pamapimod’s potential to exert beneficial therapeutic effects in rheumatoid arthritis, supporting its continued development as a promising novel treatment option.
Rheumatoid arthritis, commonly referred to as RA, is a chronic, systemic autoimmune disorder that primarily manifests as persistent inflammation of the synovial lining in multiple joints, affecting both large and small articulations in the upper and lower limbs symmetrically. The intricate pathogenesis of RA is exceptionally complex, involving a cascading activation of various synovial cell populations and the subsequent overproduction of a wide array of potent proinflammatory and destructive mediators. These include critical signaling molecules such as cytokines, lipid-derived inflammatory compounds like prostaglandins, and various proteases that directly contribute to cartilage and bone erosion. Many leading investigators in the field of rheumatology have consistently highlighted the critical and central roles played by tumor necrosis factor (TNF) and interleukin 1 (IL-1) in driving the inflammatory cascade and tissue destruction characteristic of RA pathogenesis.
Current therapeutic strategies for RA include parenteral biologic therapies, which represent a significant advancement. These sophisticated agents are designed to selectively neutralize key proinflammatory cytokines, such as TNF and IL-1, or their respective receptors. By specifically targeting these pivotal inflammatory mediators, these biologic therapies have demonstrated remarkable efficacy in substantially improving the signs and symptoms of RA, leading to a notable reduction in the number of swollen and tender joints, and, critically, slowing down the radiographic progression of erosive joint damage. However, despite these advancements and extensive research efforts, there remains a significant unmet medical need: there are currently no orally active, acceptably safe, and demonstrably effective agents on the market that primarily function to inhibit TNF or IL-1, despite numerous attempts to develop such compounds. This gap in oral therapeutic options underscores the ongoing importance of exploring novel drug targets and delivery mechanisms.
In response to this therapeutic gap, the p38 mitogen-activated protein (MAP) kinase pathway has emerged as a highly promising and intensely investigated target for the development of new oral therapies for rheumatoid arthritis. Specifically, the α-isoform of p38 is recognized as a crucial component within the intracellular signaling cascade that is indispensable for the cellular production of key proinflammatory cytokines, notably TNF-α and interleukin-1β. The p38 pathway is broadly responsive to a multitude of extracellular stimuli, encompassing various cellular stress signals such as exposure to lipopolysaccharide, osmotic stress, heat shock, and direct stimulation by the very cytokines it helps produce, including TNF-α and IL-1β. This central role in inflammation makes it an attractive therapeutic target.
Pamapimod is a selective inhibitor specifically designed to target the p38α MAP kinase isoform. This novel compound has undergone rigorous evaluation for its potential to treat the debilitating signs and symptoms of rheumatoid arthritis and for its prospective disease-modifying effects, which could alter the underlying progression of the disease. Preclinical studies have provided compelling evidence of pamapimod’s anti-inflammatory properties, demonstrating its ability to inhibit the production of TNFα, IL-1β, and/or IL-6 across a variety of nonclinical models, encompassing both in vitro cellular systems and in vivo animal models. Furthermore, it has been shown to effectively reduce inflammation in mouse models of collagen-induced arthritis, a widely used experimental model for RA.
The present study was comprehensively designed with a dual primary objective: first, to thoroughly evaluate the potential for pharmacokinetic interactions between pamapimod, the p38 mitogen-activated protein kinase inhibitor, and methotrexate (MTX) when these two agents are administered concomitantly in patients diagnosed with rheumatoid arthritis (RA). Understanding such interactions is critical for safe and effective co-administration. Second, the study aimed to assess the preliminary pharmacodynamic effects of pamapimod, providing initial insights into its biological activity and therapeutic potential within a clinical setting. To achieve these objectives, twenty-two patients suffering from RA, who were already on a stable regimen of methotrexate (administered weekly at doses ranging from 10 to 25 mg, typically on days 1 and 8 of a cycle), were meticulously randomized. Seventeen of these patients received 300 mg of pamapimod once daily, while the remaining five patients were allocated to receive a matching placebo once daily. The administration of pamapimod or placebo commenced on day 5 and continued for a period of 10 consecutive days, concluding on day 14. To facilitate detailed pharmacokinetic analysis, blood and urine samples were systematically collected at predetermined time points both before and after drug administration on crucial study days: day 1 (when only MTX was administered), day 7 (when pamapimod was administered alone after several days of continuous dosing), and notably, day 8 (when both MTX and pamapimod were coadministered).
The rigorous analysis of the collected data yielded reassuring findings. No clinically significant changes were observed in the plasma exposures, which represent the overall systemic levels of the drugs, nor in the renal clearance rates of pamapimod, methotrexate, or their respective metabolites. This consistency was evident whether the drugs were administered separately as monotherapies or concomitantly as a combination, indicating a lack of significant pharmacokinetic interaction. From a safety perspective, the combination of pamapimod (300 mg once daily) administered for 10 days alongside weekly methotrexate was generally well tolerated by the patient cohort, with no unexpected or severe adverse events reported. Furthermore, encouraging trends were observed in the pharmacodynamic effects, which reflect the drug’s impact on disease activity. Key parameters used to assess RA disease progression—namely, the tender joint count, swollen joint count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels—all generally demonstrated a decrease between day 5 (the start of pamapimod administration) and day 14 (the end of the treatment period). This suggests a beneficial trend towards reduced inflammatory activity and improved clinical signs of the disease. The collective results of this study are highly significant, suggesting that dose adjustments for either drug are not necessary when they are administered concomitantly, thereby simplifying clinical management. Moreover, the observed reduction in pharmacodynamic markers of disease activity provides preliminary evidence that pamapimod can indeed exert beneficial effects in patients with rheumatoid arthritis, supporting its continued development as a promising novel therapeutic agent.
Rheumatoid arthritis is a chronic inflammatory disorder that necessitates long-term management, and methotrexate (MTX), typically administered in oral weekly doses, stands as a cornerstone therapy for this condition. Given the potential for pamapimod to be integrated into the treatment regimen for RA, ideally in conjunction with MTX, it becomes absolutely critical to rigorously rule out any significant drug-drug interactions that could compromise either drug’s efficacy or safety profile. Furthermore, previous clinical trials involving anti-TNF inhibitors have demonstrated a rapid onset of clinical efficacy, even in relatively small treatment arms comprising approximately 20 patients. This established precedent suggested that it might be feasible to observe some meaningful pharmacodynamic activity for pamapimod even within a study of the present size and limited duration, providing early indications of its therapeutic potential.
Consequently, this study was meticulously undertaken with several key objectives. Foremost among these was to comprehensively evaluate the pharmacokinetics (PK) of both methotrexate and pamapimod when they were coadministered to patients with rheumatoid arthritis. This involved detailed analysis of their absorption, distribution, metabolism, and excretion in a combined regimen. Additionally, the study aimed to thoroughly explore the safety and tolerability profile of this combined therapeutic regimen over the study period. Finally, a critical objective was to ascertain whether the 10-day treatment with pamapimod elicited any detectable effects on objective measures of disease activity, specifically changes in swollen and tender joint counts, and the acute phase reactants such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), which serve as biomarkers of systemic inflammation.
Materials and Methods
Study Drug
Pamapimod was meticulously supplied for the study in an oral dosage form, specifically as oval-shaped tablets, each containing 100 mg of the active pharmaceutical ingredient. To maintain blinding within the study design, matching placebo tablets were also prepared, formulated with identical excipients to the active drug tablets but intentionally devoid of pamapimod. The methotrexate (MTX) and folic acid utilized in this study were not provided by the research team but were instead the patient’s existing supply, ensuring that participants continued with their established and familiar medications.
Patients
The study successfully enrolled a cohort of twenty-two patients, each of whom satisfied a stringent set of eligibility requirements designed to ensure a homogenous and relevant study population. All participants formally met the 1987 American Rheumatism Association (ARA) criteria for the definitive diagnosis of rheumatoid arthritis, ensuring consistency in patient classification. Patients were required to be within a specific age range, from 18 to 75 years inclusive, and to have a body mass index (BMI) falling between 18 and 32 kg/m², inclusive, to minimize variability due to extreme body weight. Renal function was a critical inclusion criterion, requiring a creatinine clearance greater than 50 mL/min, calculated using the widely accepted Cockcroft-Gault equation, to ensure adequate drug elimination and patient safety. Furthermore, all enrolled patients were receiving treatment on an outpatient basis and were already on stable weekly oral doses of methotrexate for a minimum of 12 weeks prior to study entry, with the last 4 weeks immediately preceding baseline requiring a precisely stable dose ranging from 10 to 20 mg/wk. Any concomitant medications for RA, such as corticosteroids (with a maximum stable dose of 10 mg/d), hydroxychloroquine, or nonsteroidal anti-inflammatory drugs (NSAIDs), also needed to have been at stable doses for at least 4 weeks prior to baseline. Finally, patients were consistently taking concomitant folic acid, with a minimum weekly dose of 5 mg, to mitigate methotrexate-related side effects.
Study Design
This investigation was structured as a randomized, placebo-controlled, observer-blind, multiple-dose study. Its primary aim was to comprehensively evaluate the pharmacokinetics (PK), safety, and tolerability of pamapimod when administered to patients with rheumatoid arthritis who were already on a stable regimen of methotrexate. The inclusion of a placebo arm was a critical design element, serving to reduce potential bias in the rigorous assessments of safety, tolerability, and the precise effect of the investigational drug on disease activity, by allowing for direct comparison against a non-active intervention. Patients were systematically randomized to receive either pamapimod or placebo in addition to their ongoing methotrexate therapy, at a ratio of 3:1, ensuring that a larger proportion of participants received the active study drug. The study was executed across four clinical centers located in diverse geographical regions, including Canada, New Zealand, and Northern Ireland, enhancing the generalizability of the findings. All aspects of the study protocol received prior approval from the respective local ethics committees, ensuring compliance with ethical research standards. Furthermore, the entire study was conducted in strict accordance with the principles of good clinical practice (GCP) and/or the Declaration of Helsinki, prioritizing whichever ethical standard provided the greater protection for the rights and well-being of the participating patients. Prior to receiving any study drug, all patients provided their fully informed consent by signing a comprehensive consent form.
Dose Administration
In the context of dose administration, seventeen patients were randomized to receive a daily oral dose of 300 mg of pamapimod once daily, while the remaining five patients were allocated to receive a matched placebo once daily. Participants continued their existing oral doses of methotrexate, ranging from 10 to 25 mg per week, specifically administered on days 1 and 8 of the study cycle. The investigational study drug, either pamapimod (provided as three 100-mg tablets) or three matching 100-mg placebo tablets, was administered consecutively for a period of 10 days, commencing on day 5 and concluding on day 14. A crucial instruction for patients was to take their assigned study medications within 10 minutes after completing breakfast, ensuring that the meal contained a moderate amount of fat, specifically with approximately 30% of the total calories derived from fat. This specific instruction was implemented to standardize absorption, as the absorption of some drugs can be influenced by dietary fat content. Notably, methotrexate and pamapimod were specifically coadministered on day 8, allowing for the direct evaluation of their pharmacokinetic interaction under combined dosing conditions.
Pharmacokinetic Assessments
To thoroughly characterize the pharmacokinetic profiles and potential interactions of the study drugs and their metabolites, a rigorous blood and urine sampling schedule was implemented. The pharmacokinetic parameters of methotrexate (MTX) and its primary metabolite, 7-hydroxy MTX, were initially evaluated on day 1, following the sole administration of MTX, serving as a baseline for its individual pharmacokinetic behavior. Subsequently, the pharmacokinetic parameters of pamapimod and its specific metabolites, RO4493992 and RO4498496, were assessed on day 7, following the sole administration of pamapimod, to characterize its individual disposition. Critically, on day 8, the pharmacokinetic parameters of both drugs, along with their respective metabolites, were concurrently evaluated following their concomitant administration, allowing for direct assessment of any drug-drug interactions. On each of these pivotal sampling days (days 1, 7, and 8), blood samples were meticulously collected at predetermined time points: immediately predose, and at 1, 2, 4, 6, 8, 12, and 24 hours postdose. Additionally, on days 1 and 8, an extra blood sample was collected at the 48-hour postdose mark to capture the full elimination profile of methotrexate. Complementing the blood sampling, all voided urine was collected over a continuous 24-hour time period, specifically from days 1 to 2 and days 8 to 9. These 24-hour urine collections were segmented into separate specimens during intervals of 0 to 6 hours, 6 to 12 hours, and 12 to 24 hours postdose, enabling the calculation of renal clearance and cumulative urinary excretion.
Disease Assessments
While there were no stringent disease activity eligibility requirements for patient enrollment, a systematic approach was employed to assess the impact of pamapimod on key parameters of rheumatoid arthritis disease status. Prior to the initiation of pamapimod dosing on day 5, which served as the baseline for this particular assessment, and again on day 14, after the completion of the 10-day treatment period with pamapimod, the disease status of each patient was meticulously evaluated. This assessment relied on a set of well-established and clinically relevant parameters: the tender joint count (TJC), which quantifies the number of joints that are painful to touch; the swollen joint count (SJC), which measures the number of joints exhibiting visible swelling; the erythrocyte sedimentation rate (ESR), a blood test that serves as a general marker of inflammation; and C-reactive protein (CRP) levels, another acute phase reactant that indicates systemic inflammation. Additionally, the Disease Activity Score 28 (DAS28) was calculated at baseline on day 5. The DAS28 is a composite score widely used in rheumatology to quantify disease activity, incorporating the TJC from 28 specified joints, the SJC from 28 specified joints, the ESR, and the Patient’s Global Health Assessment, which captures the patient’s subjective perception of their overall health status.
Safety Assessments
Throughout the entire duration of the study, a comprehensive array of safety assessments was systematically conducted to monitor patient well-being and identify any potential adverse effects associated with the study drugs. These routine safety measures included the regular recording of vital signs, such as blood pressure, heart rate, respiratory rate, and body temperature. Twelve-lead electrocardiograms (ECGs) were periodically performed to assess cardiac electrical activity and identify any potential cardiac adverse events. Extensive laboratory test results, encompassing hematology, clinical chemistry, and liver and renal function panels, were consistently monitored for any abnormalities that might indicate drug-related toxicity. All adverse events (AEs) reported by patients or observed by investigators were meticulously documented, characterized by severity, and assessed for their potential relationship to the study drugs. Furthermore, any concomitant medications being taken by the patients were thoroughly recorded to identify potential drug-drug interactions or contributing factors to observed adverse events. These rigorous safety assessments ensured a comprehensive understanding of the tolerability profile of the pamapimod and methotrexate combination.
Data Analysis
The pharmacokinetic (PK) profiles of both methotrexate and pamapimod, along with their respective metabolites, were rigorously evaluated utilizing a noncompartmental analysis approach. This established analytical method was performed using WinNonlin Pro, Version 3.2, a specialized pharmacokinetic software developed by Pharsight Corporation. From the collected plasma concentration data for both pamapimod and methotrexate, as well as their metabolites, a comprehensive set of PK parameters was derived. For pamapimod and its metabolites (RO4493992 and RO4498496), the area under the concentration-time curve from 0 to 24 hours (AUC0-24) was calculated, reflecting the total drug exposure over a 24-hour period. For methotrexate (MTX) and its metabolite (7-hydroxy MTX), the area under the concentration-time curve from 0 to infinity (AUC0-∞) was determined, providing an estimate of the total systemic exposure over an infinite time horizon. Additional critical PK parameters calculated for both drugs included the maximum observed plasma concentration (Cmax), indicating the peak drug level reached in the bloodstream; the terminal half-life (t1/2), which estimates the time required for the drug concentration to decrease by half during the elimination phase; the time to maximum observed plasma concentration (tmax), representing the time at which Cmax is achieved; the apparent clearance (CL/F), an estimate of the volume of plasma cleared of drug per unit time; and the apparent volume of distribution (V/F), which indicates the extent of drug distribution into body tissues. Furthermore, for pamapimod, the AUC0-24 ratios of its metabolites to the parent drug were calculated to assess the extent of metabolite formation. For methotrexate and 7-hydroxy MTX, additional PK parameters were determined from the urine samples. These included the cumulative amount of drug excreted in urine over the 0- to 24-hour interval (Ae0-24), the percentage of drug excreted in urine over 24 hours (%Ae0-24), and the renal clearance (CLr), which specifically quantifies the rate at which the drug is cleared by the kidneys. It is important to note that no urine PK analysis was performed for pamapimod due to the absence of an adequately sensitive assay for its detection in urine.
To precisely evaluate the effect of methotrexate coadministration on the systemic exposure of pamapimod and its metabolites, a two-way analysis of variance (ANOVA) model was applied. This statistical model incorporated factors for individual patient variability and the specific treatment condition (pamapimod alone versus pamapimod coadministered with MTX) and was performed on logarithmically transformed values of AUC0-24 and Cmax, utilizing data collected from days 7 and 8 exclusively from patients randomized to the pamapimod group. Conversely, to assess the effect of pamapimod coadministration on the systemic exposure of methotrexate and its metabolite 7-hydroxy MTX, a different ANOVA model was employed. This model included factors for treatment sequence, patient variability within each sequence, and the specific treatment condition (MTX alone, MTX + placebo, and MTX + pamapimod). This analysis was conducted on logarithmically transformed values of dose-normalized AUC0-∞ and Cmax, using data from days 1 and 8 from all study patients. From these ANOVA models, least squares means and their corresponding 90% confidence limits were derived. These values were then exponentiated to provide estimates of the mean exposure ratios and their corresponding 90% confidence limits on the original concentration scale, allowing for a direct and clinically meaningful interpretation of any potential interactions.
Disease activity parameters were systematically listed and descriptively summarized to provide an overview of the patient population and observed trends. The Disease Activity Score 28 (DAS28) was meticulously calculated for each patient at baseline on day 5 using a well-established standard equation. This equation is defined as: DAS28 = 0.56 × √28TJC + 0.28 × √28SJC + 0.7 × ln ESR + 0.014 × GH. In this formula, “28TJC” represents the tender joint count observed in 28 specified joints, “28SJC” denotes the swollen joint count in those same 28 joints, “ln ESR” refers to the natural logarithm of the erythrocyte sedimentation rate, and “GH” signifies the patient’s global assessment of their health, typically measured on a 100-mm visual analog scale (VAS). The sample size for the study was carefully determined to ensure statistical power for the primary pharmacokinetic objective. A target enrollment of 15 patients receiving the pamapimod + MTX combination was chosen to ensure that the 90% confidence interval (CI) for the mean exposure ratio would be contained within the conventionally accepted bioequivalence interval of 80% to 125% when comparing coadministration versus separate administration of pamapimod and MTX. This calculation was based on the assumption that no significant drug-drug interaction would occur and also factored in an estimated 19% within-patient coefficient of variation (CV) for the 24-hour AUC of methotrexate, derived from previous data. The specific number of patients allocated to the placebo group was determined based on clinical judgment, balancing the need for a control arm with logistical and ethical considerations for patient exposure to placebo.
Analytical Techniques
The precise and quantitative analysis of all plasma and urine samples, crucial for determining the concentrations of pamapimod and its two specific metabolites (RO4493992 and RO4498496), as well as methotrexate (MTX) and its primary metabolite, 7-hydroxy MTX, was expertly conducted by Covance Bioanalytical Services, a specialized contract research organization. For the analysis of pamapimod, RO4493992, and RO4498496, along with the internal standard essential for accurate quantification, the compounds were first extracted from the biological samples using a robust protein precipitation method involving acetonitrile. Following this extraction, the resulting supernatant, now largely free of interfering proteins, was carefully diluted with a solution of 0.1% formic acid. The prepared samples were then analyzed using a meticulously validated liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) method. This highly sensitive and selective analytical technique allows for the precise separation of compounds and their subsequent detection and quantification. The standard curve, which defines the quantifiable range for these compounds, was established to span from 5.00 nanograms per milliliter (representing the lower limit of quantitation, LLQ) up to 5000 nanograms per milliliter for pamapimod, RO4493992, and RO4498496. This was achieved using a relatively small plasma sample volume of 0.100 milliliter, optimizing sample conservation.
For the analysis of methotrexate and its metabolite 7-hydroxy MTX, along with their internal standard, a separate and equally validated extraction method was employed for human plasma samples, also relying on protein precipitation. After this initial precipitation, the supernatant was carefully collected and evaporated under a gentle stream of nitrogen gas, concentrating the analytes. The dried residue was then reconstituted in a suitable solvent and subsequently analyzed using another distinct and thoroughly validated LC/MS/MS method, specifically optimized for MTX and its metabolite. The standard curve for MTX and 7-hydroxy MTX in plasma ranged from 1.00 nanogram per milliliter (the LLQ) to 250 nanograms per milliliter, utilizing a plasma sample volume of 0.0500 milliliter. For the analysis of methotrexate, 7-hydroxy MTX, and their internal standard from human urine samples, a third distinct and validated LC/MS/MS method was developed. The extraction process for urine involved solid-phase extraction, a technique known for its effectiveness in cleaning up complex biological matrices. The extracted analytes were then analyzed. The standard curve for MTX and 7-hydroxy MTX in urine ranged from 1.00 nanogram per milliliter (the LLQ) to 250 nanograms per milliliter, using a urine sample volume of 0.200 milliliter. Throughout all these bioanalytical procedures, the precision and accuracy results for the calibration standards consistently remained well within predefined acceptance criteria, ensuring the high quality and reliability of the quantitative data.
To ensure the utmost consistency and comparability of C-reactive protein (CRP) results across all patient samples, a crucial acute phase reactant, all analyses were performed at a single, dedicated laboratory. A high-sensitivity assay, specifically conducted with the IMMULITE 1000 system (manufactured by Pathway Diagnostics Corporation), was utilized. This approach minimized inter-laboratory variability and provided precise measurements of CRP, reflecting systemic inflammation.
Results
Demographic Data and Baseline Characteristics
The demographic and baseline characteristics of the study patient cohort were meticulously documented to ensure representativeness and enable appropriate interpretation of results. The majority of the enrolled patients were female, comprising 18 out of 22 individuals (82%), and predominantly of white ethnicity, accounting for 20 out of 22 participants (91%). The remaining patients included one individual of Asian descent (4.5%) and one of Egyptian origin (4.5%). The mean age of the patient population was approximately 52 years, with individual ages ranging from 34 to 68 years, and notably, the age distribution was similar between the pamapimod and placebo treatment groups. Regarding anthropometric data, patients randomized to the pamapimod group exhibited a mean ± standard deviation (SD) body mass index (BMI) of 29.0 ± 5.5 kg/m², while those in the placebo group had a mean ± SD BMI of 25.8 ± 3.1 kg/m². The weekly methotrexate (MTX) dose administered to patients ranged from 10 to 25 mg, with an average dose of 15 mg per week consistently observed across both treatment groups. At day 5, prior to the initial administration of pamapimod or placebo, the Disease Activity Score 28 (DAS28) values, a comprehensive measure of rheumatoid arthritis activity, spanned a range from 1.9 to 6.4. Within this range, four patients demonstrated a DAS28 score below 2.6, indicating a state of clinical remission, while seven patients exhibited a DAS28 score of 4.5 or greater, signifying significant disease activity. Importantly, the overall baseline disease activity, as reflected by DAS28 scores, was comparable between the two treatment groups at the point of randomization.
Of the total 22 patients initially enrolled in the study, two were unfortunately withdrawn prematurely. Both of these patients had been randomized to receive pamapimod 300 mg once daily. One patient’s withdrawal, which occurred on day 2 after receiving a single dose of MTX and no pamapimod, was due to a violation of the entry criteria. The second patient was withdrawn on day 6 for administrative reasons, having received one dose of MTX and two 300-mg doses of pamapimod prior to withdrawal. These withdrawals were documented and considered in the final analyses, but did not significantly impact the overall study conclusions due to the sufficient remaining sample size.
Pharmacokinetics Results
Effect of Methotrexate on the Pharmacokinetics of Pamapimod and Its Metabolites
Out of the 17 patients initially randomized to receive pamapimod treatment, 15 were ultimately included in the robust pharmacokinetic analysis for pamapimod and its two primary metabolites. Following the oral administration of pamapimod as a single agent on day 7, pharmacokinetic data revealed that, on average, the maximum plasma concentration (Cmax) of pamapimod was achieved approximately 2.1 hours postdose, as indicated by the time to maximum concentration (tmax) (Figure 1 and Table I). The overall exposure to pamapimod, quantified by the area under the concentration-time curve from 0 to 24 hours (AUC0-24), was 21.9 micrograms per hour per milliliter. The apparent clearance (CL/F) of pamapimod was calculated to be 14.2 liters per hour, and its apparent volume of distribution (V/F) was estimated at 83.6 liters. Crucially, on day 8, when pamapimod was coadministered with methotrexate (MTX), the mean plasma concentration-time profiles for pamapimod and its two metabolites (RO4493992 and RO4498496) showed a remarkable overlap with the profiles observed on day 7, when pamapimod was administered alone (Figure 1). This visual concordance was further supported by quantitative analyses, demonstrating that the key pharmacokinetic parameters for pamapimod and its metabolites were highly comparable, irrespective of whether MTX was coadministered. The AUC0-24 and Cmax ratios for the pamapimod metabolites, RO4493992 and RO4498496, were consistently similar to those for the parent compound pamapimod itself, both when administered as monotherapy and in combination with MTX.
Detailed statistical analysis confirmed that no clinically significant difference was observed in the AUC0-24 of pamapimod when it was administered concomitantly with MTX, as compared to its administration alone. The mean exposure ratio was calculated to be 0.99, with a narrow 90% confidence interval (CI) of 0.94-1.04 (Table I). This falls well within the conventional bioequivalence acceptance criteria of 0.80 to 1.25, indicating an absence of a significant interaction on overall exposure. However, a subtle but statistically significant observation was made regarding the Cmax for pamapimod, which was approximately 19% lower (mean exposure ratio = 0.81; 90% CI = 0.69-0.95) when coadministered with MTX. While statistically significant, this modest reduction in peak concentration is generally not considered to be clinically relevant for drugs like pamapimod, particularly given the lack of impact on overall AUC.
Effect of Pamapimod on the Pharmacokinetics of Methotrexate
When methotrexate (MTX) was administered concomitantly with pamapimod on day 8, its plasma exposures, encompassing both peak concentrations and overall systemic levels, were found to be remarkably similar to those observed when MTX was administered alone on day 1 (Table II and Figure 2). Specifically, the geometric mean ratio of the dose-normalized AUC0-∞ for MTX, comparing its administration with versus without pamapimod, was calculated to be 1.12, with a 90% confidence interval of 1.05 to 1.20. Similarly, the geometric mean ratio of the dose-normalized Cmax for MTX, under the same comparative conditions, was 1.00, with a 90% confidence interval of 0.93 to 1.06. Crucially, both of these 90% confidence intervals fell comfortably within the generally accepted “no-effect” boundaries of 0.80 to 1.25, indicating that pamapimod does not exert a clinically significant impact on the systemic exposure of methotrexate. Furthermore, the renal clearance of MTX and the percentage of the MTX dose recovered in urine demonstrated consistent results, irrespective of whether MTX was administered alone or in combination with pamapimod (Table II). The mean exposure ratios (comparing with versus without pamapimod) were 0.90 for renal clearance and 1.00 for the percentage of dose recovered in urine, further confirming the lack of a significant interaction at the level of renal elimination.
However, a slight but notable increase was observed in the AUC0-∞ and Cmax values for 7-hydroxy MTX, the primary metabolite of methotrexate, when MTX was administered concomitantly with pamapimod compared to MTX alone. On average, the AUC0-∞ of 7-hydroxy MTX increased by approximately 26%, and its Cmax increased by 20% (Table II). Despite these observed increases in the plasma exposures of the metabolite, the renal clearance and the percentage of the dose recovered in urine for 7-hydroxy MTX appeared to be similar in magnitude whether administered with or without pamapimod. Given that the pharmacological activity of 7-hydroxy MTX is estimated to be only approximately 10% of that of the parent drug, MTX, the observed 20% to 26% increase in the exposure to this metabolite is not considered to be clinically relevant or to have any significant impact on the overall therapeutic efficacy or toxicity profile.
Exploratory Results for Disease Activity
The study also included exploratory assessments of disease activity parameters to gain preliminary insights into pamapimod’s pharmacodynamic effects. Swollen Joint Count (SJC), Tender Joint Count (TJC), Erythrocyte Sedimentation Rate (ESR), and C-reactive protein (CRP) were assessed for 21 patients at baseline (prior to the first pamapimod dose on day 5) and for 20 patients prior to dosing on day 14. A noteworthy trend emerged when comparing the treatment groups: the group means for all four markers of disease activity—SJC, TJC, ESR, and CRP—demonstrated a consistent decline for patients who received both methotrexate and pamapimod. This suggests a beneficial impact of pamapimod on reducing inflammatory markers and clinical signs of RA. In contrast, among patients receiving methotrexate and placebo, only the SJC showed a decrease in its mean value, while CRP and ESR did not show a clear beneficial trend, and TJC actually showed a slight increase (Table III). These exploratory findings, while not powered for definitive efficacy conclusions, suggest a positive pharmacodynamic effect of pamapimod in reducing disease activity markers in patients with rheumatoid arthritis.
Safety Results
Adverse Events
Throughout the course of the study, a total of 76 adverse events (AEs) were reported by 20 of the enrolled patients. The vast majority of these adverse events were rated by the investigators as mild in severity, and notably, none of the reported AEs were considered severe. The overall event rate, calculated as the ratio of reported events to the number of patients, was found to be similar between the placebo-treated group and the pamapimod-treated group, indicating a comparable overall safety profile. Dizziness and headache emerged as the most frequently reported adverse events across the study population. Specifically, 4 out of 5 patients in the placebo group and 8 out of 16 patients in the pamapimod group experienced either dizziness, headache, or both after day 5, which marked the initiation of pamapimod or placebo administration. Within the pamapimod group, seven patients specifically reported dizziness, with four of these individuals experiencing multiple episodes, collectively accounting for 18 out of the total 21 reported dizziness events. The majority of these dizziness episodes (19 out of 21) were characterized as mild. It was observed that dizziness in patients experiencing multiple episodes tended to occur within a few hours of drug dosing and typically resolved spontaneously within a timeframe ranging from 11 minutes to 3.5 hours. In the placebo group, two patients reported dizziness, but neither experienced multiple episodes.
Additionally, five patients in the pamapimod group experienced an adverse event that was classified as an infection. These infections included folliculitis, reported in three patients, and other mild infections such as nasopharyngitis and a urinary tract infection (UTI). Only one patient required specific treatment for an infection; this individual had a pre-existing, asymptomatic UTI that persisted without complication throughout the trial and was administered amoxicillin and clavulanic acid. These infection rates were monitored closely to assess any potential immunosuppressive effects of pamapimod.
Vital Signs and ECGs
Throughout the entire study duration, rigorous monitoring of vital signs and 12-lead electrocardiograms (ECGs) was performed for all patients. Importantly, no clinically relevant or significant changes were observed in any of the vital signs parameters (e.g., blood pressure, heart rate) or the ECG parameters (e.g., QTc interval, PR interval), indicating that pamapimod and its combination with methotrexate did not induce any notable cardiovascular effects.
Laboratory Tests
In terms of laboratory test results, four patients in the pamapimod group exhibited alanine aminotransferase (ALT) values that were above the upper limit of normal (defined as >30 U/L). Interestingly, two of these patients already had mildly elevated ALT values of 31 U/L at the time of screening prior to study entry. The peak elevations in ALT levels among these four patients occurred approximately 2 days after receiving both methotrexate and pamapimod concurrently, specifically on day 10, with values ranging from 35 to 81 U/L. Subsequent to this peak, the ALT values consistently declined in all four affected patients, and by day 21, their levels were either at or near normal, with the exception of one patient. Importantly, no ALT elevations were observed in any patient belonging to the placebo group. Furthermore, aspartate aminotransferase (AST) values for all patients remained consistently within normal limits throughout the entire study period, providing additional reassurance regarding hepatic safety.
Discussion
This study unequivocally demonstrated that pamapimod and methotrexate (MTX), when administered concomitantly, do not exhibit a clinically significant drug-drug interaction. This finding is of considerable practical importance, as it provides crucial data that facilitated the design and conduct of subsequent phase II studies involving pamapimod. Specifically, the results of this interaction trial allowed the phase II studies to proceed without the necessity of adjusting the background methotrexate dose or pamapimod dosage when the two compounds were coadministered, simplifying clinical trial logistics and potentially future patient management. Overall, the drug combination was found to be well tolerated by the study participants. However, it was observed that patients treated with pamapimod experienced a higher frequency of dizziness and infections compared to the placebo-treated patients, suggesting that these may be potential adverse effects associated with pamapimod. Encouragingly, the measured joint counts and acute phase reactants, which are objective indicators of disease activity in rheumatoid arthritis, showed a discernible diminution in pamapimod-treated patients when compared to those receiving placebo. This observation strongly suggests that pamapimod might indeed be efficacious in larger, adequately powered clinical trials specifically designed to assess therapeutic efficacy.
Regarding the pharmacokinetic observations, the area under the concentration-time curve (AUC) values for pamapimod and its metabolites remained essentially unaltered in the presence of methotrexate, indicating that overall systemic exposure to pamapimod is not significantly affected by co-administration. There was, however, a slight reduction, approximately 20%, in the maximum observed plasma concentration (Cmax) for pamapimod and its metabolites when administered concurrently with methotrexate. The study protocol specified that the investigational drug (300 mg pamapimod) and methotrexate were administered to patients simultaneously, specifically within 10 minutes after the completion of breakfast. While a precise mechanism for this modest Cmax decrease is not definitively known, it is hypothesized that there might be some form of competitive absorption between the two drugs in the gastrointestinal tract. Nevertheless, this modest decrease in Cmax is generally not considered to be clinically significant, particularly in the context of the unchanged overall exposure (AUC), which is often a more relevant indicator of therapeutic effect for many drugs.
For methotrexate and its primary metabolite, 7-hydroxy MTX, both the plasma and urine exposures were found to be highly consistent with those previously reported in existing literature for this patient population, especially when considering oral doses ranging from 10 to 25 mg per week. This consistency validates the study’s pharmacokinetic methods for MTX. It was observed that there appeared to be a modest increase, specifically ranging from 20% to 26%, in the plasma AUC and Cmax for 7-hydroxy MTX in patients who received pamapimod concurrently. Despite this observed increase in the exposure to the metabolite, it is crucial to consider its pharmacological activity. Given that the activity of 7-hydroxy MTX is estimated to be only approximately 10% of that of the parent compound, methotrexate, the impact of a 20% to 26% increase in exposure to this less potent metabolite is not considered to be clinically relevant or to significantly alter the overall therapeutic or toxicity profile of the methotrexate regimen.
Subsequent to the completion of this foundational drug-drug interaction study, pamapimod progressed into two pivotal phase IIB clinical studies. These larger trials collectively enrolled over 400 patients and involved dosing for a duration of 12 weeks. The reassuring results from the present study, particularly concerning the lack of a clinically significant drug-drug interaction, provided critical support for the design of these phase II studies, allowing the combination therapy to be conducted without the need for adjusting the established methotrexate or pamapimod dose levels. While pamapimod demonstrated some efficacy, its effects were ultimately deemed weak when compared to placebo in one study and were less efficacious than methotrexate in an active comparator trial. Interestingly, the decreases observed in C-reactive protein (CRP), swollen joint count (SJC), and tender joint count (TJC) in this drug-drug interaction trial were, in part, recapitulated in the phase IIB studies. Specifically, acute phase reactants like CRP, along with SJC and TJC, showed an initial decline during the first two weeks of pamapimod dosing, often in a dose-dependent manner. However, a crucial observation was that these beneficial pharmacodynamic effects tended to return toward baseline values over the subsequent 10 weeks of the study’s dosing period. Similar patterns of initial response followed by a return towards baseline have also been documented with other p38 inhibitors, suggesting a potential class effect. This implies that the pharmacodynamic effects, while present, do not persist over a longer 12-week dosing period. This important finding suggests that future proof-of-concept trials for novel agents in rheumatoid arthritis should be designed with a treatment duration significantly longer than just two weeks, to accurately rule out phenomena such as tachyphylaxis, where the body’s response to a drug gradually diminishes with repeated administration, as potentially seen with pamapimod.
Furthermore, this initial drug-drug interaction trial proved remarkably predictive of the adverse event (AE) profile that subsequently emerged in the larger phase II clinical trials for pamapimod. In those larger studies, adverse events such as dizziness, various infections, and elevations in alanine aminotransferase (ALT) levels were consistently observed at a higher incidence in pamapimod-treated patients compared to those receiving placebo or methotrexate alone. These specific adverse events are also typical of those commonly seen with other p38 inhibitors, suggesting that they are likely class effects. This conclusion is further strengthened by the fact that the chemical scaffolding, or core molecular structure, is distinct for pamapimod when compared to other p38 inhibitors like SCIO469 and VX-702. The consistent emergence of these side effects across chemically distinct compounds targeting the same pathway strongly supports their classification as class-specific adverse reactions, providing valuable insight for future drug development in this therapeutic area.