
==== Front
Clin Transl Sci
Clin Transl Sci
10.1111/(ISSN)1752-8062
CTS
Clinical and Translational Science
1752-8054
1752-8062
John Wiley and Sons Inc. Hoboken

10.1111/cts.13908
CTS13908
CTS-2024-0127
Dei
Article
Article
Bioavailability of a novel sustained‐release pellet formulation of 5‐flucytosine in healthy‐fed participants for use in patients with cryptococcal meningitis
Short course treatment for Cryptococcal meningitis
Ibnou Zekri Lassout et al.
Ibnou Zekri Lassout Nabila https://orcid.org/0009-0003-5554-0362
1
Goyal Vishal https://orcid.org/0000-0003-3564-129X
2 vgoyal@dndi.org

Krantz Edrich 3
Simon Francois https://orcid.org/0009-0003-6068-9874
1
Neven Anouk https://orcid.org/0000-0003-2253-0427
4
Eriksson Johanna https://orcid.org/0000-0002-9921-0969
5
Saayman Amaria 3
Satam Vijay 6
Ruffell Carol https://orcid.org/0000-0002-3180-7574
7
Victor Sarika 3
Chenel Marylore 5
Celebic Aljosa https://orcid.org/0009-0001-0614-6874
4
Caplain Henri https://orcid.org/0009-0008-6977-326X
1
Gillon Jean‐Yves https://orcid.org/0000-0003-1467-0403
1
Deshmukh Abhijit 8
Antarkar Amit 8
Sjögren Eric 5
Ribeiro Isabela https://orcid.org/0000-0001-7402-4628
1
1 Drugs for Neglected Diseases Initiative Geneva Switzerland
2 Drugs for Neglected Diseases Initiative New York New York USA
3 FARMOVS Bloemfontein South Africa
4 Competence Center for Methodology and Statistics (CCMS) Luxembourg Institute of Health Strassen Luxembourg
5 Pharmetheus Uppsala Sweden
6 Drugs for Neglected Diseases initiative New Delhi India
7 DNDi GARDP Southern Africa Cape Town South Africa
8 Viatris Hyderabad India
* Correspondence
Vishal Goyal, Drugs for Neglected Diseases Initiative, 40 Rector Street, 16th Floor, New York, NY 10006, USA.
Email: vgoyal@dndi.org

18 9 2024
9 2024
17 9 10.1111/cts.v17.9 e1390823 7 2024
27 3 2024
25 7 2024
© 2024 Farmovs (Pty) Ltd, Luxembourg Institute of Health, Mylan Laboratories Ltd, Pharmetheus AB, Drugs for Neglected Diseases Initiative, DNDi GARDP Southern Africa NPC. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Abstract

Cryptococcal meningoencephalitis (CM) is an opportunistic fungal infection and a major cause of death among people living with human immunodeficiency virus in sub‐Saharan Africa. 5‐flucytosine (5‐FC) is a unique, brain‐permeable antifungal agent used to reduce mortality from CM and to prevent disease in individuals carrying cryptococcal antigen. 5‐FC has a short plasma half‐life, requiring 6‐hourly oral dosing with an immediate‐release (IR) formulation, a significant challenge in hospital and outpatient settings, risking a lack of compliance. We recently reported the relative bioavailability in fasting conditions of a sustained release (SR) oral pellet formulation of 5‐FC. In this phase I study, we assessed the safety and pharmacokinetic profiles of the new 5‐FC SR formulation in a single dose (2 × 3000 mg), relative to 5‐FC IR tablets (Ancotil®; 1500 mg b.i.d.) in healthy participants in fed conditions. This randomized, two‐period crossover study was conducted in South Africa to confirm the dose of the identified 5‐FC SR formulation for a twice‐daily 5‐FC regimen in patients. Thirty‐six healthy participants were included. All treatments were well tolerated and no serious adverse event was reported. C max and AUC(0–t) for the SR formulation (49.2 ± 10.49 μg/mL and 640.4 ± 126.4 h.μg/mL, respectively) were significantly higher than for the IR formulation (36.8 ± 7.61 μg/mL and 456.6 ± 72.8 h.μg/mL, respectively). A physiological based pharmacokinetic model (PBPK) predicted that under fasting conditions, 6000 mg SR pellets would show a good overlap with the IR product (3000 mg b.i.d), thus 6000 mg SR 5‐FC b.i.d. in fasting conditions is recommended.

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cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:18.09.2024
Ibnou Zekri Lassout N , Goyal V , Krantz E , et al. Bioavailability of a novel sustained‐release pellet formulation of 5‐flucytosine in healthy‐fed participants for use in patients with cryptococcal meningitis. Clin Transl Sci. 2024;17 :e13908. doi:10.1111/cts.13908
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pmc Study Highlights

WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

5‐flucytosine is a key, brain‐permeable antifungal for the treatment of cryptococcal meningoencephalitis; however, it has a limited plasma half‐life and needs to be administered every 6 h, posing major practical challenges that may result in treatment failure and death. WHAT QUESTION DID THIS STUDY ADDRESS?

This phase I study assessed the bioavailability of a sustained‐release formulation of 5‐flucytosine after single‐dose oral administration, relative to an immediate‐release formulation of 5‐flucytosine in healthy, fed participants using a randomized two‐period crossover study. The treatments were evaluated for safety and their pharmacokinetic profiles. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

Both treatments were well tolerated and had a similar safety profile. The primary PK parameters Cmax and AUC(0–t) from the 5‐flucytosine SR formulation were significantly higher than from the 5‐flucytosine IR tablets, and the geometric mean ratios were above the conventional bioequivalence limits. The median T max was reached earlier for the 5‐flucytosine SR pellets. The 5‐fluorouracil (5‐flucytosine metabolite) exposure was too low for proper PK assessment. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

Our data suggests that the sustained‐release formulation of 5‐flucytosine identified in the previous phase I study is a good candidate for further investigation in future studies in patients. Our physiologically‐based pharmacokinetic modeling predicts that a 6000 mg dose of that formulation, given twice a day in the fasted state, has the potential to produce C trough and C max values that are effective and safe.

INTRODUCTION

An estimated 112,000–181,000 people die each year from cryptococcal meningoencephalitis (CM), an opportunistic infection, prevalent primarily in low‐ and middle‐income countries (LMICs). 1 , 2 Most (>80%) patients are immunosuppressed due to inadequately managed HIV infection. 3 Cryptococcal meningitis is one of the leading cause of HIV‐related deaths. 4

The 2022 World Health Organization (WHO) guidelines for treatment of CM 5 include treatment with an initial single dose of liposomal amphotericin B (10 mg/kg), 14 days of 5‐flucytosine (5‐FC, Ancotil®) (100 mg/kg/day divided into four doses per day), and fluconazole (1200 mg/daily for adults). 5 Combination treatment with fluconazole and amphotericin B aims to reduce the potential for treatment failure and development of fungi resistant to 5‐FC. 5‐FC is also a key component of the alternative induction regimens, having been shown to increase survival versus fluconazole, as a partner drug to amphotericin B deoxycholate. 6

5‐FC is an old therapeutic agent with unique antifungal properties associated with high bioavailability that allows it to cross the blood–brain barrier. 7 The antifungal capacity of 5‐FC is due to its metabolism by the fungus into 5‐fluorouracil (5‐FU), which is further converted to metabolites that inhibit fungal RNA and DNA synthesis. 8 5‐FC has broad‐spectrum antifungal properties, including activity against Candida, Aspergillus, Cladosporium, and Cryptococcus. Drug exposure, however, must be carefully controlled to manage CM, as 5‐FC’ short plasma half‐life (3–4 h) makes this a challenge. 8 The current regimen with the immediate‐release (IR) tablets includes drug administration every 6 h, with reports of missed doses, notably in overburdened, resource‐limited settings, 9 , 10 and hence, a potential impact on efficacy. Additionally, because many patients with CM have a reduced consciousness level, standard 5‐FC IR tablets must be crushed for four times daily administration by nasogastric tube, even though these tablets have not been approved for this administration route.

A consortium of European and African partners was set up to develop a new 5‐FC sustained‐release (SR) formulation that is to be widely accessible, safe, affordable, easy to use, suitable for nasogastric administration and that would contribute to reducing the mortality caused by CM. A candidate SR formulation was selected for further investigations following a recent phase I study, 11 and was evaluated under conditions reaching the therapeutic dose in the additional phase I study described here.

In the aforementioned first study, 11 three SR 5‐FC pellet formulations delivering 5‐FC over 12 h and with three different release rates were assessed for their safety and pharmacokinetic (PK) profiles as a single dose (1 × 3000 mg at 0 h), relative to commercial IR tablets (3 × 500 mg at 0 h and 3 × 500 mg at 6 h) in healthy, fasted participants. All treatments were well tolerated. The C max and AUC(0−t) values were significantly lower for the three SR formulations than for the IR tablets at the same total dose of 3000 mg (highest geometric mean ratio was 49.0% (90% CI: 43.5–55.1%) for C max and 53.6% (90% CI: 49.2–58.3%) for AUC(0−t)). The SR prototype with the higher plasma exposure was selected for further clinical evaluation.

Based on in vitro dissolution profile and data from the first phase I study, physiologically‐based PK (PBPK) modeling suggested a higher dose (6000 mg) of this prototype is needed under fasting and fed conditions for healthy participants to meet target therapeutic 5‐FC plasma exposure (20–100 μg/mL).

In this phase I clinical study, we expected to confirm, in fed condition that the SR formulation triggers 5‐FC plasma exposure between the therapeutic boundaries (≥20 and ≤100 μg/mL), and above the minimum inhibitory concentration (MIC) capable of inhibiting Cryptococcus spp. fungal growth by ≥90% (MIC90) of 4.0–8.0 μg/mL. The European Medicines Agency “Guideline on the pharmacokinetic and clinical evaluation of modified release dosage forms” acknowledges that in general, modified‐release formulations are not bioequivalent to their IR form and they are developed based on the rationale that there is a relationship between the pharmacodynamic response and the characteristics of the systemic exposure to the active substance. 12 This study compared the PK profile in healthy participants in the fed condition of a single oral dose of the SR formulation with that of the commercial 5‐FC IR tablet used previously as reference, 11 and (a) confirm that clinical development of the SR pellets could be pursued and (b) evaluate the new modeled dosage regimen. Given the unknown prandial status of some unconscious patients at the time of hospitalization for the management of CM, assessing dosing in fed conditions was important.

METHODS

Objectives, participants, and oversight

This open‐label, bioanalytical laboratory‐blind, randomized, two‐period crossover study with orally administered 5‐FC IR tablets and 5‐FC SR pellets was conducted in healthy males and females in fed condition between November 2022 and May 2023 at a single study center (FARMOVS Clinical Research Organization, Pharmacology Building, University of the Free State, Bloemfontein in South Africa). The primary objective was to compare the bioavailability of a single oral dose (2 × 3000 mg at 0 h) of 5‐FC SR pellets (Formulation D of the previous phase I study) relative to 5‐FC IR tablets (the reference product; Mylan SAS (now Viatris), USA) (3 × 500 mg at 0 h and 3 × 500 mg at 6 h after first dosing). The SR formulation of 5‐FC was developed to release in vitro 5‐FC as a percentage of the nominal dose at rates of not more than 45% at 1 h, 50%–80% at 3 h, and not less than 85% at 9 h. The qualitative and quantitative composition of 5‐FC SR formulation is presented in Table S7 and the in vitro dissolution data are presented in Table S8. The 6000 mg dose for SR treatment was proposed based on the results from the PBPK model developed previously and the PK data in fasted condition (Figure 1a), 11 in order to meet the targeted therapeutic 5‐FC concentrations (20–100 μg/mL) in plasma. These therapeutic 5‐FC levels were based on previous studies that showed that (a) MIC90 is 4.0–8.0 μg/mL, suggesting plasma 5‐FC concentrations ≥20 μg/mL reach efficacy and (b) 5‐FC concentrations >100 μg/mL are likely to be toxic. 13 , 14

FIGURE 1 Simulated and observed 5‐FC plasma concentration–time profiles in (a) fasted and (b) fed state. Immediate‐release formulation dosing was 1500 mg at t = 0 h and t = 6 h and sustained‐release formulation dosing was 3000 mg at t = 0 h. Observations from the 35 enrolled patients in the first phase I study are represented by dots. The solid line and shaded area represent the simulated geometric mean and the 5–95% quantiles of the PBPK model for a virtual population (n = 200). Below limits of quantification (BLQ) values removed from the plot.

The secondary objective of the study was to evaluate the safety and tolerability of the two 5‐FC formulations. For inclusion and exclusion criteria see Text S1.

Due to the narrow therapeutic index of 5‐FC and the expected plasma exposure, stopping criteria were set to minimize the risk to healthy participants in the study. Details on the screening period, washout periods, and follow‐up are provided in Figure 2.

FIGURE 2 Study flow chart. Each participant received the two treatments (5‐FC immediate release and 5‐FC sustained release), with washout periods (crossover design), in randomized order. IMP, investigational medicinal product, PK, pharmacokinetic.

Ethical considerations

This study was conducted in compliance with the Declaration of Helsinki, the International Council for Harmonization Good Clinical Practice guidelines, and the South African Good Clinical Practice guidelines. The clinical study protocol, including participant information sheets and informed consent forms, and protocol amendment No. 1, were reviewed by an independent ethics committee and by the South African Health Products Regulatory Authority (SAHPRA). This study was registered at the Pan African Clinical Trial Registry (Trial ID: PACTR202211836109661) before enrolling the first participant.

Randomization and study treatment

Participants were randomly assigned in a 1:1 ratio to their treatment sequence before the first Investigational Medicinal Product (IMP) administration using block randomization with a block size of 4. The randomization schedule was provided by the Luxembourg Institute of Health, Competence Center for Methodology and Statistics and was generated utilizing the PROC PLAN procedure of SAS® software.

In this study, during the two test periods, participants received each of the following treatments once: Reference: Flucytosine 500 mg IR tablets (total dose of 3000 mg, administered as two doses: 3 × 500 mg [0 h] and 3 × 500 mg [6 h])

Test: Flucytosine 3000  mg SR pellets (total dose of 6000 mg, administered as a single dose: 2 × 3000  mg [0 h])

Safety and tolerability

An independent safety and data monitoring committee (DMC) was established before the start of this study, which reviewed safety data on an ongoing basis.

As a precautionary measure, four participants were initially enrolled, dosed for the two treatment periods, and monitored. The DMC reviewed emerging safety data from these four participants after the poststudy visit had been completed and concluded that it was safe to continue with the dosing of the remaining 32 participants.

An interim safety analysis was performed on safety data and selected PK samples after all participants had completed PK sampling in treatment period 1; the results were reviewed by the DMC for any safety concerns before participants continued to treatment period 2.

Bioanalytical methods

These are detailed in our previous publication. 11

Pharmacokinetic evaluations

The PK population included all participants who completed the PK sampling in both periods, for whom primary PK parameters could be calculated for both treatment periods, and who had no major protocol deviations thought to impact the PK analysis.

PK parameters were processed using Analyst® version 1.6.3 for the acquisition of raw data, and Watson LIMS™ version 7.4.2 for the management and processing of laboratory information.

The software packages have been validated according to the requirements of Part 11 of Title 21 of the Code of Federal Regulations of the United States. 15 , 16

Calculation of the PK parameters was performed using non‐compartmental analyses with Phoenix® WinNonlin® 8.3 and the actual sampling time intervals relative to the IMP administration. The parameters AUC(0–t) and AUC(0–inf) were calculated using the linear up/log down method. T 1/2 was calculated as 0.693/lλz. Concentration values reported as “below the limit of quantification” (BLQ), that is, <0.4 μg/mL, were set to 0 at predose and before the first quantifiable concentration, and to “missing” after the last quantifiable concentration. Not reportable concentrations were set to missing.

Statistical analysis

Sample size considerations were driven by the primary PK evaluation and the planned comparison between the test and reference treatments. Assuming the intra‐participant coefficient of variation (CV) to be no more than 28% (based on the previous phase I study data 11 ), with 30 participants in total (i.e., 15 participants per sequence), a crossover design had 80% power to reject both the null hypothesis that the ratio of the test mean to the standard mean was below 0.8 and the null hypothesis that the same ratio was above 1.25 (i.e., that the test and standard were not equivalent), in favor of the alternative hypothesis (the means of the two treatments were equivalent, assuming that the expected ratio of means was 1, the crossover ANOVA MSE (in scale) was 0.28 (the standard deviation [SD] differences, σ (ln scale) was 0.396), that data were analyzed in the natural log scale using t‐tests for differences in means, and that each t‐test was made at the 5% level). To account for possible drop‐outs and withdrawals, recruitment of 36 participants was planned.

The plasma concentrations of 5‐FC and the PK parameters were summarized in the PK population using descriptive statistics (n, arithmetic mean, standard deviation (SD), CV% of arithmetic mean, median, minimum, maximum, and geometric mean).

The PK outcomes C max, AUC(0–t), and AUC(0–inf) were log‐transformed and analyzed based on a linear mixed effects model with sequence, product, and period as fixed effects and participant as a random effect. The residual variance from the model was used to construct 90% confidence intervals (CIs) for least‐squares (LS) mean differences between treatments. These differences were then back‐transformed to obtain point estimates (ratios) of geometric means and corresponding 90% CIs. T max, t 1/2, and λz were analyzed nonparametrically using the Wilcoxon signed‐rank test based on the median of differences between treatments across participants.

All statistical analyses were performed using the SAS System Version 9.4 (SAS Institute, Cary, NC, USA).

Physiologically‐based pharmacokinetic (PBPK) modeling

PBPK modeling was performed in PK‐Sim® v. 9.1. The PBPK model for 5‐FC was initially developed based on literature data. The legacy model included a description of oral absorption of an IR formulation (Ancobon®) as immediately dissolved (i.e., oral solution) and subsequent permeation across the intestinal mucosa, elimination via glomerular filtration, and fraction unbound of 97%. It was used to support the SR prototype design. 17 The legacy PBPK model was used as a starting point for further model development. The observed PK data in the fasted state (from the first phase I study with the SR prototypes 11 ) was used to refine the legacy model, by optimizing selected model parameters, including Weibull functions for each SR formulation. Further, to simulate the 5‐FC PK after intake of food, the gastric emptying time was prolonged in accordance with the available implementation in PK‐Sim®. Increased solubility as a result of food intake was not considered for 5‐FC, as it is a water‐soluble drug, with high solubility, and not expected to be affected by solubilization by bile salts. Prediction of food effect indicates increased AUC and C max in the fed state compared with fasted state (about a 20% increase), see Table 1. Thereafter, the observed PK data in the fed state (present study) were used to construct a final model in the fed state by optimizing selected model parameters. The models were evaluated by overlaying the simulated concentration–time profiles with observed data and comparing exposure parameters to assess the ability to describe the clinical data. Details on the PBPK model are provided elsewhere. 11 , 18

TABLE 1 Simulated median flucytosine (5‐FC) plasma maximum concentration at steady state (Css, max) and percentages of participants in the population above the 100 mg/L threshold (upper limit of targeted therapeutic 5‐FC concentrations), stratified by food intake. Values are presented as median (5 and 95 percentiles).

	Median (5th–95th perc.)	Proportion > 100 microg/mL	
Sustained release 5‐FC fasted	54.6 (32.8–93.8)	4%	
Sustained release 5‐FC fed	66.0 (40.7–122)	15.5%	

The PBPK model was used to guide further development and suggest a dosage within the therapeutic interval for patients with CM.

RESULTS

Participants

Participant disposition is summarized in Table S1; 35/36 participants completed the study. A demographic summary of all 36 participants is shown in Table S2. While the number is small, the participant population, included both genders (30 males and 6 females), and the country's major racial groups (32 were Black, 1 Caucasian, 1 Colored and 2 were of Mixed race).

One participant was withdrawn from the study by the investigator after a positive drugs of abuse test on admission to treatment period 2. A list and details of protocol deviations can be found in the supplement.

Safety

All 36 participants enrolled were included in the safety analysis.

Six (16.7%) of the 36 participants reported a total of 9 treatment‐emergent adverse events (TEAEs) during the study. Eight (89%) out of the 9 reported events were of mild intensity and 1 (11%) event was of moderate intensity. Nervous system disorders and eye disorders were the most common categories of TEAE, with 3 participants reporting headache, 1 reporting eye pain, and 1 reporting eye pruritus and vision blurred. Headache is a common AE in healthy participants of phase I clinical trials. Two participants were administered concomitant medications, both for the reported TEAEs. Three of these events were considered by the investigator as possibly related to the IMP (one event related to the reference product and two events related to the test product, see also Tables S3, S4, and S6). IMP‐related TEAEs (three events) were single events of headache (mild severity), palpitations (mild severity), and transaminases increase (moderate severity). No SAEs or deaths were reported during this study and none of the TEAEs were severe.

During clinical chemistry evaluations at 30 h postdose with the reference product, it was observed that one participant had an isolated and transient elevated AST concentration of 137 U/L (3.0 times the ULN, upper limit of normal) and ALT of 98 U/L (1.7 × ULN) without other associated liver abnormalities, that reversed to normal values 9 days after the last dose. This was reported as an AE of moderate intensity considered related to the reference product.

There were no clinically significant electrocardiogram abnormalities, changes in vital signs, or physical findings after administration of the IMPs. No case of leukopenia or thrombocytopenia was observed.

5‐FC and 5‐FU pharmacokinetic profiles

All 35 completers were included in the PK analysis. A total of 0.7% of the PK samples (included in the analysis) were taken outside the pre‐specified tolerance windows and were reported as protocol deviations. No participant was excluded from the PK population due to major protocol deviations. Only values at predose (100%), 30 min (1.4%), 36 h (1.4%), and at 48 h (30%) postdose were below the limit of quantification (BLQ).

The individual 5‐FC PK profiles and the arithmetic mean graphs for each treatment formulation are represented jointly in Figure 3. Table 2 represents the summary statistics for the PK parameters and further illustrates the differences in PK behavior between the IR and SR formulations.

FIGURE 3 Flucytosine (5‐FC) plasma concentrations for the 5‐FC immediate release (IR) tablets (reference product) (two doses, 1500 mg each, 6 h apart) and the 5‐FC sustained release (SR) formulations (one dose of 6000 mg) in individual participants. The red line represents the geometric mean.

TABLE 2 Summary of plasma flucytosine (5‐FC) pharmacokinetic parameters (PK population).

Parameter (unit)	Arithmetic Mean ± SD	
Reference (IR 5‐FC)	Test (SR 5‐FC)	
n	N = 35	n	N = 35	
C max (μg/mL)	35	36.8 ± 7.61	35	49.2 ± 10.49	
AUC(0–t) (h μg/mL)	35	456.6 ± 72.79	35	640.4 ± 126.41	
AUC(0–∞) (h μg/mL)	35	463.7 ± 73.06	35	661.5 ± 130.38	
λ z (/h)	35	0.11 ± 0.02	35	0.08 ± 0.03	
t ½ (h)	35	6.2 ± 0.87	35	9.4 ± 4.48	
t max (h) (median and range)	35	8.0 (1.0–12.0)	35	6.0 (3.0–7.0)	
Note: Number of participants in the PK population is 35. The reference product is 500 mg flucytosine IR tablets, Test product is the sustained‐release formulation of 5‐FC.

Abbreviations: IR, immediate release 5‐FC; n, number of participants assessed; SD, standard deviation; SR, sustained release 5‐FC.

Table S5 shows the results obtained from the linear mixed modeling. The 90% CIs for the primary PK parameters (C max and AUC(0–t )) for 5‐FC were above the conventional acceptance range (i.e., 80%–125%) for establishing comparable bioavailability between the test SR formulation and IR reference product. When comparing SR pellets with IR tablets, the geometric mean ratio was 133.6% (90% CI: 124.2–143.7) for C max and 139.3% (90% CI: 132.4–146.5) for AUC(0–t), respectively. With regards to the secondary PK end point, AUC(0–inf), geometric mean ratios were also above the conventional acceptance range. The median T max was significantly higher with the reference product (p‐value < 0.0001). Lambda_z was also higher, and t 1/2 was lower, with the reference product than with the SR formulations.

PK data on 5‐FU were used as supportive data. As 95.7% of the plasma 5‐FU values were BLQ, no summary statistics have been provided for this 5‐FC metabolite. The maximum concentration detected in plasma was 10.9 ng/mL.

Physiologically‐based pharmacokinetic modeling

Based on the data from this trial and simulated 5‐FC plasma concentration–time profiles in fed condition (see Figure 1b), the suggested 6000 mg b.i.d. dose for the SR formulation will potentially exceed the therapeutic interval, compared with a dose regimen of 1500 mg four times a day for the 5‐FC IR formulation. Some of the observed 5‐FC SR plasma concentration data were above the predicted 5‐FC plasma concentration, probably due to the variability of drug absorption for the weight of the participants. Simulating PK parameters at steady state in fasted condition, with weight‐based dosing of 100 mg/kg/day (divided between four dosing occasions/day) for the IR formulation and 6000 mg b.i.d. for the SR formulation, and their grouping by body weight (Figure 4), showed that for weights below 60 kg, most IR and SR data overlapped, even if some participants receiving the SR formulation reached plasma concentrations >100 μg/mL at C max. Patients with a higher weight tended to be overexposed when given an IR weight‐adjusted dose. Based on that most patients living with advanced HIV and diagnosed with CM have a body weight of 40–60 kg, a PK simulation was conducted in fed and fasted conditions for participants of 60 kg with an IR formulation dosing of 1500 mg 4 times per day, and SR formulation dosing of 6000 mg b.i.d. (Figure 5). Based on these data, the SR formulation should be administered at 6000 mg b.i.d. in fasting conditions to avoid overexposure. Furthermore, the C trough after the modeled first dose (e.g., at 12 h) for some participants administered the SR formulation was either at or below the lower therapeutic level of 20 microg/mL. As described previously, MIC90 for 5‐FC was determined to be 4.0–8.0 μg/mL. In this simulation, after the first dose, the 5th percentile of simulated C trough is slightly below the MIC90 of 8 microg/mL, but above 4 mg/L. From the second administration of the SR, the 5th percentile of simulated C trough is above the MIC90 of 8 microg/mL. Most of the virtual patients receiving the SR formulation exceeded the MIC90 (97.2%), as well as the lower therapeutic threshold (62.7%). Moreover, 56.2% of participants who received the SR formulation had a C trough at 12 h that was less than 1.5 the interquartile range of the C trough for participants who received the IR formulation.

FIGURE 4 Estimated PK parameters at steady state in fasted conditions for an immediate‐release formulation and sustained‐released formulation in all participants grouped by body weight. Weight‐based dosing is 100 mg/kg/day (divided into four dosing occasions/day) for the immediate‐release formulation and sustained‐release formulation dosing is 6000 mg b.i.d. MIC90 is represented by the dotted line. The boxplots show the median represented by the central line in the box, the box edges represent the 25th and 75th percentile, the lower and upper whiskers represent the smallest and largest values within 1.5 times the interquartile range, respectively, and the dots represent the outliers as defined by >1.5 times or <3 times the interquartile range.

FIGURE 5 Simulated 5‐FC plasma concentration–time profiles up to steady state in fasted and fed state for a participant of 60 kg. Black (immediate‐release treatment) and colored (sustained‐release treatment) lines and shaded areas represent the median and 5%–95% range of the simulations, respectively. Immediate‐release formulation dosing was 1500 mg four times per day and sustained‐release formulation dosing was 6000 mg b.i.d. The dashed lines represent the therapeutic interval and the dotted line represents MIC90.

Given the therapeutic interval of 5‐FC, the simulation ranges in Figure 1b suggest that some virtual participants administered the SR formulation could exceed the upper therapeutic limit (e.g., 100 microg/mL) in the fed group, and to a lesser extent, in the fasted group.

DISCUSSION

In this study, we aimed to assess the relative bioavailability of a single dose of a new 5‐FC SR formulation, compared with two doses of a 5‐FC IR tablets administered 6 h apart. The aim was to achieve with the two formulations, 5‐FC plasma exposure above the MIC90 (PD target) and below the safety threshold (≤100 μg/mL), 19 in fed condition.

Overall, 36 participants were enrolled and equally randomized to the two sequence (S) arms (S1 = SR‐IR, S2 = IR‐SR). The two arms had similar demographic characteristics, with a majority being male (88.9% in S1 and 77.8% in S2) and Black (88.9% in S1 and S2), an overall mean age of 30 years (mean of 27.7 years in S1 and 32.2 in S2), and an overall mean body mass index of 24.5 kg/m2 (25.0 in S1 and 24.1 in S2).

In general, both IMPs were well tolerated and there was no signal of drug‐induced liver toxicity or bone marrow depression. No deaths or SAEs were reported, and all AEs were of mild or moderate intensity. One participant presented with a transient elevation of AST and ALT (considered related to the reference product).

Conversion of 5‐FC to 5‐FU has been proposed as one key mechanism of the development of 5‐FC‐associated toxicity. 20 The 5‐FU blood concentrations measured in this study (with a highly sensitive method) were significantly lower than earlier reports following administration of 5‐FC, 13 and in line with our observations in the previous study. 11 This is in line with the absence of additional safety or tolerability events with the new 5‐FC SR formulation, compared 5‐FC IR.

In vitro, the time to the release 100% flucytosine is less than 1 h with the IR tablet, whereas it is more than 9 h with the SR formulation (Table S8). The release profiles of the two formulations are significantly different. Therefore, comparison of their similarity by generating f2 values was not deemed useful. The bioavailability of 5‐FC from the SR pellets exceeded the bioavailability of 5‐FC from the Ancotil® IR tablets at the doses administered in the study, in fed condition. The mean AUC(0–t) was 1.4‐fold higher for the SR product than for the IR product. Similarly, the mean C max was 1.3‐fold higher for the SR product than for the IR product. This increase in bioavailability is possibly due to a longer transit time following ingestion of a meal (with a longer time period for dissolution in the stomach and subsequent increased absorption to occur in the small intestine) and potential increased dispersion/agitation/mixing. The PBPK model was updated with the data from this phase I study for the prediction of exposure at steady state after administration of the SR formulation at 6000 mg in fasted condition, matched with the predicted exposure after administration of the IR formulation at 100 mg/kg/day in patients with a weight below 60 kg. PBPK modeling confirmed that SR 5‐FC administration in fed condition may result in exposures at steady state exceeding safety boundaries, with a recommended 5‐FC SR regimen in participants with HIV‐associated CM at 6000 mg b.i.d., in fasting condition (with suggested intake before breakfast and before dinner, at least 4 h after the last meal). The unknown feeding status of unconscious patients at hospital admission should not pose any risk of exceeding the therapeutic interval, as the C max of the first dose is below the upper therapeutic limit of 100 μg/mL.

A higher peak concentration and a higher exposure were observed with the 6000 mg SR formulation dose compared with the 2 × 1500 mg IR formulation dose in fed condition. Repeated dosing was not feasible in healthy participants, thus PBPK was used as a tool to optimize the dose regimens and predict PK profile in patient populations. The PBPK model was used to first support the selection of an SR formulation among 3 prototypes 11 and then to further support dosing and formulation selection in food conditions. The legacy PBPK model for 5‐FC was developed based on literature data related to an IR formulation. Then the PK study from the fasted phase I study was used to further update and refine the legacy model. 18 Similar PK parameters for the IR formulation (weight‐based dosing, 100 mg/kg/day) and SR formulations (6000 mg b.i.d.) were predicted in the low (40–60 kg) weight‐group, which represents the large majority of the targeted population of adults living with HIV‐associated CM. The results of this phase I study will inform the dose selection for a phase II study, assessing the safety, efficacy, and PK of the new 5‐FC SR formulation in patients with CM in the African region. In addition to the oral route, this new SR formulation has the potential to be used more easily for nasogastric administration and also for self‐administration by outpatients. In the phase II study, the palatability and the acceptability of the SR formulation by both patients and health workers will also be investigated, as well as the potential economic impact of substituting the IR tablets by the SR pellets formulation.

AUTHOR CONTRIBUTIONS

NIZL, VG, EK, FS, AN, JE, AS, VS, CR, SV, MC, AC, HC, JYG, AD, AA, ES, and IR wrote the manuscript. EK, VG, AN, AC, AJS, HC, JYG, and IR designed the research. VG, EK, NIZL, AN, AS, ML, AC, JYG, and IR performed the research. NIZL, VG, EK, AN, JE, AS, MC, AC, HC, JYG, ES, and IR analyzed the data. AS, VA, JE, MC, AD, and AA contributed new reagents/analytical tools.

FUNDING INFORMATION

This project (grant RIA2018CO‐2516) is part of the European and Developing Countries Clinical Trials Partnership Association (EDCTP2) program supported by the European Union, with additional funding from the Swiss Agency for Development and Cooperation (SDC), Switzerland; Médecins Sans Frontières International, and other private foundations and individuals.

CONFLICT OF INTEREST STATEMENT

The authors declared no competing interests for this work.

Supporting information

Table S1

Table S2

Table S3

Table S4

Table S5

Table S6

Table S7

Table S8

Appendix S1

ACKNOWLEDGMENTS

We thank Dr Louise Burrows (Drugs for Neglected Diseases initiative) for help with editing and formatting.

DATA AVAILABILITY STATEMENT

The data underlying the results of this study are available upon request because they contain potentially sensitive personal information, which must be deidentified at the individual level. Interested researchers may contact the Drugs for Neglected Diseases initiative (DNDi), commissioner of this study, for data access requests via email at ctdata@dndi.org. Researchers may also request data by completing the form available at https://www.dndi.org/category/clinical‐trials/. In this, they confirm that they will share data and results with DNDi and will publish any results open access.
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