Category: 3. Business

Switzerland’s federal prosecutor has filed charges against the failed bank Credit Suisse and its new owner, UBS, over the long-running “tuna bonds” loan scandal that crashed Mozambique’s economy nearly a decade ago.

The Swiss attorney general said on Monday that it had brought money-laundering charges against an unnamed employee of Credit Suisse, but was also taking action against the lender and its rival-turned-owner UBS.

The attorney general’s office accused the banks of “organisational deficiencies” that ultimately failed to prevent wrongdoing and meant the suspicious transactions were not reported until 2019, after the US Department of Justice announced it was launching its own criminal proceedings.

The prosecutor added: “In 2016, in particular, considerable defects existed in the companies’ risk management, compliance and internal directives systems in connection with combating money laundering.”

UBS took over Credit Suisse as part of an emergency rescue deal brokered by Swiss authorities in 2023. UBS said: “We firmly reject the office of the attorney general’s conclusions and will vigorously defend our position.”

The tuna bonds scandal arose from $2bn (£1.5bn) worth of loans that Credit Suisse arranged for the Republic of Mozambique between 2013 and 2016. The loans were said to be going to government-sponsored investment schemes including maritime security projects and a state tuna fishery, located in the south-east African country’s capital, Maputo.

However, a portion of the funds went unaccounted for, with one of Mozambique’s contractors later found to have secretly arranged “significant kickbacks” worth at least $137m, including $50m for bankers at Credit Suisse, meant to secure more favourable deals on the loans, according to the Financial Conduct Authority.

The scam snowballed and eventually caused the International Monetary Fund to suspend its assistance to Mozambique, leading to a crash in the country’s economy.

Credit Suisse had already settled the case with US and UK regulators in 2021, having paid $275m to American watchdogs and £147m to Britain’s Financial Conduct Authority.

UBS also subsequently agreed to settle the case with Mozambique in October 2023, shortly before a trial was due to kick off in London courts. Mozambique had been pursuing $1.5bn in damages over economic losses after the IMF and international donors pulled their financial support.

The Swiss attorney general’s office accused Credit Suisse and its owner of “not taking all the required and reasonable organisational measures in the relevant period in 2016 to prevent the money laundering that was allegedly committed”.

Credit Suisse was sold to UBS in an emergency deal in March 2023, when customers started to pull money from the lender amid a mini-banking crisis that primarily affected US lenders but later spread to Zurich.

Credit Suisse had for years been mired in scandals, but panic over its future grew after its largest shareholder, Saudi National Bank, ruled out any extra funding for the Swiss lender despite the growing turmoil.

The crisis of confidence eventually forced Swiss authorities to offer emergency loans to Credit Suisse, before eventually orchestrating a shotgun takeover by Switzerland’s largest bank, UBS, which bought the lender for a cut price of 3bn Swiss francs. It left UBS handling a raft of legacy scandals from its former rival.

Minimal residual disease (MRD) assessment and circulating tumor DNA (ctDNA) monitoring are emerging as important tools that may refine clinical management in lymphoma. In an interview with OncLive®, Sarah Rutherford, MD, discussed how ctDNA is beginning to demonstrate meaningful clinical value, particularly in diffuse large B-cell lymphoma (DLBCL), where post-treatment PET scans frequently pose interpretive challenges. Rutherford noted that ctDNA may help clinicians distinguish between true residual disease and inflammatory findings, improving confidence in both surveillance and follow-up decision-making.^1,2

She highlighted that much of the foundational progress in MRD and ctDNA technology has come from the solid tumor field, where these assays are already widely used. That experience, she explained, has helped establish reimbursement pathways and clinical familiarity that may ease the transition into hematologic practice. Recent studies evaluating ctDNA at baseline and end-of-treatment in DLBCL have shown its prognostic relevance, supporting its potential role in risk stratification and monitoring.

Rutherford also pointed to the personalized, tumor-informed Signatera assay (Natera), one of the few ctDNA platforms currently available for clinical use. By leveraging patient-specific tumor sequencing and noninvasive blood sampling, the assay enables individualized ctDNA tracking that may inform surveillance and, eventually, guide therapeutic intervention. As next-generation sequencing and ctDNA-based MRD testing continue to advance in parallel, Rutherford emphasized that these modalities are likely to become increasingly integrated into risk assessment, treatment tailoring, and long-term disease monitoring for patients with lymphoma.

Rutherford is an associate professor of clinical medicine in the Division of Hematology/Oncology at Weill Cornell Medicine in New York, New York.

OncLive: What is currently understood about MRD and ctDNA and their potential role in the management of lymphoma?

Rutherford: As a community, we are so excited that ctDNA is starting to be used more in these diseases, especially DLBCL. There are difficult situations within the management of patients in which we’re not sure exactly how to interpret PET scans, for example, and I believe ctDNA is going to be a great tool to help us guide patients along their journey from diagnosis to treatment and follow-up.

Based on the data in solid tumors, what lessons from ctDNA and MRD research have informed approaches in lymphoma?

It is interesting that there are more advancements, in some ways, in this area of monitoring in solid tumors compared to hematologic malignancies. What I have been most concerned about, from a practical standpoint, is making sure the patients aren’t getting bills related to this type of testing. The fact that it’s already being done in solid tumors [paves] a way that, I hope, will lead to reimbursement, so that we can be checking this and feel confident that the patients won’t get a bill for it.

What are some of the current approaches for measuring ctDNA and MRD within patients with lymphoma?

ctDNA in lymphoma is being [assessed] in clinical trials and not as much in clinical practice, although I believe we’re on the brink of that changing. There have been some compelling studies published and reported over the last couple years. One is the DIRECT study [NCT04226937] that was done in the UK, showing both at baseline and after treatment that the ctDNA amount is prognostic for progression-free survival. [Additionally], data that came from the HOVON group that were reported at ASCO this past year that I found to be the most useful showed what the ctDNA results are at the end of treatment, for example, in DLBCL.

Sometimes we have a PET scan that’s hard to interpret; we’re not sure exactly what to make of it. ctDNA testing will help us feel more confident monitoring someone—even if they have a positive PET scan at the end of treatment—but we know their ctDNA was negative. On the flip side, if someone has positive ctDNA, then I believe we’re going to be much more cautious about the monitoring and their follow-up from there.

At this point most of us are not using ctDNA to make decisions quite yet, but we’re close to being able to do that. I know there’s a clinical trial being done in patients who have ctDNA positivity at the end of treatment for DLBCL that is putting them potentially onto a novel therapy, such as CAR T-cell therapy.

Where I see it most useful in the current state is in monitoring patients. Over the years, studies looking at surveillance PET scans have shown that if someone’s in a complete response after treatment for DLBCL, it’s never been shown to improve overall survival to do lots of PET scans. There are negative aspects [such as] radiation exposure, cost, and anxiety. How I would first use ctDNA testing in DLBCL would be in those surveillance situations. When they come in for their 3-month follow-up visit, we’ll feel reassured if we get a result back showing their ctDNA is undetectable. However, if we find ctDNA is detectable, then we can do a PET scan, assess, potentially do a biopsy, and figure out if they need another line of therapy.

Focusing on Signatera assay specifically, how is the assay designed, and what potential advantages does it offer compared with other MRD testing methods?

The Signatera [assay] is one of the only available assays for ctDNA to be used in the clinical setting. Many of the assays are research-based and aren’t available for us to use. I work at an academic institution, and we have familiarity as a group because this company—the company that makes the Signatera assay—is very well established in solid tumor malignancies. My understanding is that they create a personalized and tumor-informed assay for each person based on their tumor tissue that then can be tracked using a noninvasive strategy of collecting peripheral blood for ctDNA analysis.

They’re ramping up in terms of collecting data in DLBCL. There have been some studies already showing that it can be predictive of outcomes and may enable surveillance, and also potentially intervention. For example, if someone has positive findings at the end of treatment, perhaps eventually we’ll have a clear-cut way of managing those patients so that we can optimize their outcomes.

What remaining hurdles need to be overcome before they can be integrated into routine practice?

It will be helpful to have it done at baseline so that it’s clear—because the tumor tissue is needed to develop this personalized assay, I believe we should be sending the test at baseline so that we can have that information available. One of the biggest hurdles is just ensuring insurance company acceptance and payment for this.

I also believe that it is a little challenging to know what to do with the information right now. At end of treatment, if patients have positive ctDNA and I don’t have a clinical trial available that would potentially treat a patient in that situation, it’s hard to treat them based on that information. So, more clinical trial information about what to do in that situation is a hurdle. And again, making sure it’s covered by insurance is a key part. But if I could ensure that, I would be comfortable using it at baseline and then in the follow-up monitoring setting to really augment our clinical data and hopefully avoid unnecessary imaging studies.

In the context of both established and emerging biomarkers, how do you envision ctDNA and MRD integrating into risk assessment and treatment decision-making?

One of the key other tests we’re starting to do more in DLBCL is next-generation sequencing [NGS] panels. In some of the other diseases, particularly hematologic malignancies, we have already been doing NGS for years, but it hasn’t been that effective yet in terms of giving us different strategies for how to treat [patients with] DLBCL.

But there are a couple groups who have created genetic clusters and algorithms. One is the DLBCL classifier developed by the Harvard group, where you can input mutation information and it helps figure out the genetic subtype a patient has. At this point, we don’t treat patients differently based on that information, but there is a clinical trial through the National Clinical Trials Network being planned where patients will be randomized to different treatments based on their genetic cluster.

I see this field moving forward with both next-generation sequencing and ctDNA/MRD approaches hand-in-hand, so that we can tailor treatment strategies—not just the type of therapy but also the length of treatment and monitoring in remission. Both will make a big difference in our patients’ lives in the coming years.

For those who are still becoming familiar with MRD and ctDNA in lymphoma, what is your key take-home message about their potential impact on the treatment paradigm?

We’ve heard the term MRD for years, and there were different assays used previously, such as chromosome-based assays. These newer technologies for ctDNA analysis are likely more accurate and will be more accessible. It’s important for us to look at the clinical trials being done, learn from those, and decide together as a field how we’re going to use this in practice so that we can tailor treatment correctly for patients.

References

1. Zhang S, Wang X, Yang Z, et al. Minimal residual disease detection in lymphoma: methods, procedures and clinical significance. Front Immunol. 2024;15:1430070. doi:10.3389/fimmu.2024.1430070
2. Rossi D. Assessing remission in diffuse large B-cell lymphoma: will minimal residual disease add value to positron emission tomography? J Clin Oncol. 2025;43(34):3631-3635. doi:10.1200/jco-25-01932

Introduction

Background

Multiple sclerosis (MS) is a chronic, immune-mediated neurological condition affecting mostly young individuals worldwide with a variety of clinical manifestations and trajectories, which can lead to permanent disability as it evolves.

MS among Asians is relatively less prevalent compared to Caucasians. Recent meta-analyses reported the incidence and prevalence in Asian and Oceanic regions being 2.4/100,000, and 37.9/100,000, respectively,¹ whereas the incidence and prevalence in Europe were 2/100,000 and 59/100,000, respectively.² Local data were sparse with the latest study more than 10 years ago estimated the prevalence at 4.8/100,000.³

Current evidence suggested that the pathological hallmark of MS featured demyelination,⁴ which is caused by T-lymphocytes, and with growing evidence, B-lymphocytes⁵ as well, though the exact mechanism was still largely unknown. Several demographic and environmental factors including smoking, vitamin D deficiency, Epstein-Barr virus (EBV) infection were recognised to be associated with MS and its prognosis.

McDonald criteria were used for the diagnosis of MS since 2001.⁶ According to the updated 2017 McDonald criteria (Supplementary Table 1),⁷ the diagnosis of MS requires at least one typical clinical attack with evidence of demyelination in the central nervous system, and dissemination in time and space, without other plausible alternative diagnosis.

An attack typically includes optic neuritis, transverse myelitis, brainstem, and cerebellar syndrome.⁷ When it occurred in an isolated manner, it was termed clinically isolated syndrome (CIS). In relapsing-remitting MS, the patients have multiple attacks but remain stable in-between. On the other hand, in progressive MS, there exists progressive worsening of disability, with initial remitting relapsing phase present in SPMS and absent in PPMS.⁸

In the recent 30 years, a growing number of DMTs have become available. While none of them can completely cure MS, these were shown to decrease the clinical relapses, and to postpone the progression of this debilitating illness.⁹

The first DMT was Interferons beta (IFN), approved in the USA and Europe in 1993 and 1995, respectively.¹⁰ The mechanisms of action were immunomodulatory, for instance, decreasing T cell activation and inflammatory cytokines production.¹¹ Azathioprine was a popular therapeutic option historically in MS before specific therapies became widely available as it inhibits purine synthesis and induces T cells apoptosis as a purine analogue.¹²

Fingolimod (FTY) is the first oral DMT used to treat RRMS. It is a sphingosine 1-phosphate receptor modulator,¹³ which was shown to influence the mobilisation of lymphocytes, exhibiting its immunomodulatory effect.¹⁴ More specific therapies emerged with time, including dimethyl fumarate (DMF) and teriflunomide (TFN). DMF exerts its cytoprotective effect in the central nervous system,¹⁵ while TFN inhibits B and T cell proliferation through inhibition of dihydro-orotate dehydrogenase,¹⁶ which is an enzyme used in pyrimidine synthesis for lymphocytes production.

The scientific development of various monoclonal antibodies opened the door further for the newer treatment options. One example is alemtuzumab which acts on CD-52 to deplete T and B cells from circulation and has been shown to provide an excellent treatment response.¹⁷

In Hong Kong, IFNs were the first approved therapies for MS since 2012,¹⁸ while the oral DMF and TFN later became popular choices as first line DMTs. In general, FTY remained the second line DMT. When the previously used DMTs were ineffective, use of high efficacy DMTs, for example, alemtuzumab, natalizumab, and cladribine, was considered. This study would like to focus on the more commonly used DMTs in Hong Kong so as to reflect the local treatment practice for our MS patients.

Aims and Objectives

Aim

To date, there were only few observational cohort studies in Hong Kong comparing the efficacy, tolerability and discontinuation pattern of the rapidly growing number of DMTs, and the prognostic factors for local MS patients. Our study aims to provide real-world data on the use of DMT in one of the tertiary hospitals for managing MS patients in HK.

Primary Objectives

To assess the effectiveness, tolerability and discontinuation pattern of various DMTs.

Secondary Objectives

(1) To explore baseline characteristics of MS patients in Hong Kong.

(2) To determine factors leading to DMTs discontinuation.

(3) To assess prognostic factors for MS in terms of worsening of Kurtzke’s Expanded Disability Status Score (EDSS)¹⁹ (Supplementary Table 2) and annualised relapse rate (ARR).

Method and Material

Inclusion and Exclusion Criteria

Patients with a diagnosis of MS given by the attending clinician were identified by the Clinical Data Analysis and Reporting System. Adult patients aged over 18 years, who had follow-up visits in the Queen Elizabeth Hospital neurology specialist outpatient clinics during the period between 1st December 2012 and 30th November 2022 were included in our study. The exclusion criteria included alternative diagnoses to MS made by clinicians, and unconfirmed diagnosis of MS.

Study Design

This was a retrospective observational study using data from clinical records or electronic patient record via the Clinical Management System after approval by the ethics committee. Various demographic and clinical parameters were retrieved for further analysis (Table 1).

Demographic factors, including age, ethnicity, smoking status and medical co-morbidities, were either self-reported by the patient or entered by the clinicians. The types of MS were identified by the attending clinician at diagnosis and in the latest visit.

At initial presentation, date of symptom onset reported by patients, date of diagnosis, initial type of symptoms, lesions in magnetic resonance imaging (MRI), baseline Kurtzke’s Expanded Disability Status Score (EDSS) were assessed. The presence of Oligoclonal bands (OCBs), and the data in white blood cells and protein level in cerebrospinal fluid (CSF) were analysed for the patients who underwent lumbar puncture. Moreover, the presence of abnormalities in visual (VEP), somatosensory (SSEP), and brainstem auditory (BAEP) evoked potentials, were noted.

A relapse was defined as, a new symptom which lasted for more than 24 hours, in the absence of concurrent illness. An MRI activity was defined as either new or gadolinium contrast-enhancing lesions in the subsequent MRI scans. The study period was from the date of symptom onset, till the censor date, 30th November 2022 or death, whichever earlier.

The annualised relapse rate (ARR) was calculated by dividing the number of relapses by the study period in years. For comparison, ARR 2 years prior to commencement of DMT was recorded. The presence of disability progression with EDSS worsening was also evaluated. It was defined by increase in EDSS score of at least 0.5 point if the baseline EDSS was greater than 5.5, of 1 point if the baseline EDSS was within 1 to 5.5, or 1.5 point if baseline EDSS was 0.

The number of DMT uses, the duration between diagnosis and commencement of DMTs, was recorded in patients who commenced DMTs. Adverse effects by the patient or the attending physician were reported. The duration of DMT was defined as the first use of DMT till discontinuation. Discontinuation of DMT, as defined by the interruption of treatment for 60 days or more, was also evaluated.

Statistical Methods

Statistical analyses were conducted using Statistical Package for Social Sciences (SPSS) version 26. Descriptive statistics were used for patients’ demographic and clinical factors. Percentage was used for categorical data, while mean and standard deviation (SD) were calculated for continuous data respectively. Kaplan-Meier curve was used for the DMT discontinuation with log rank test used between various DMT subgroups, where Cox regression was followed if statistical significance detected. Fisher’s exact test was used to study the relationship between EDSS worsening and various factors. Independent samples t-test were used to study the relationship between ARR and various factors, respectively. A p-value of less than 0.05 was considered statistically significant. Formal power calculation was not feasible due to the retrospective nature of the study and discussed this as a limitation.

Ethic Issues and Confidentiality

The study was approved by the Research Ethics Committee in Kowloon Central and East Cluster of Hospital Authority. Individual patient was assigned with a unique code for identification purpose. Individual patient consent was waived as this was a retrospective medical record review using anonymised data. Patient confidentiality was maintained throughout the study, in accordance with the Declaration of Helsinki.

Results

Of 125 identified patients via Clinical Data Analysis and Reporting System, an electronic health database operated by the Hospital Authority of Hong Kong, 22 were excluded where 18 (17.4%) of them had alternative diagnoses including Neuromyelitis optica spectrum disorder (NMOSD), acute disseminated encephalomyelitis, unconfirmed diagnosis, and 4 (3.2%) due to irretrievable or missing clinical information, leaving 103 patients were included in the study with the mean follow-up period 191.1 (range 14–794, SD 136) months.

Patient Characteristics

Demographic Factors

In the study cohort of 103 patients, 74 (71.8%) and 29 (28.2%) patients were female and male respectively, giving the female:male ratio 2.55:1. The vast majority were of Chinese ethnicity (99, 95.2%). Eighty-three (80.6%) patients were non-smoker while 14 (13.6%) of them were ever-smokers at diagnosis. The mean ages of symptom onset and diagnosis were 29.7 (SD 10.1) and 33.2 (SD 10.4), respectively (Figure 1).

Figure 1 A pie chart of distribution of disease onset across five age groups: 1–10 years (n=3, 3%), 11–20 years (n=17, 17%), 21–30 years (n=34, 35%), 31–40 years (n=30, 31%), and greater than 40 years (n=14, 14%).

Clinical Factors

Fifty-four (52.4%) patients were associated with other medical co-morbidities, which included hypertension, diabetes mellitus, hyperlipidaemia, eczema, fatty liver, thyroid disease. None of the patients had family history of MS.

The clinical characteristics of the patients is listed in Table 2. The most common type of MS at initial diagnosis was RRMS, with 69 (67.0%) patients, followed by CIS (16, 15.5%), PPMS (6, 5.83%) and SPMS (9, 8.74%) respectively. The types of 3 (2.91%) patients at diagnosis were untraceable. In 83 (80.6%) of the patients, the baseline EDSS were either recorded or extrapolated with most commonly lies below 3.5 (74, 71.9%).

The commonest types of signs and symptoms at initial presentation were presented as paresthesia (38, 36.9%), followed by visual symptoms (30, 29.1%) and limb weakness (29, 28.2%), with the rest in Table 2. Among the 88 (85.4%) patients with clear period of symptom onset and diagnosis, the mean duration between symptom onset and diagnosis was 36.8 (range 0–262, SD 54.3) months.

All except 1 (0.97%) patient had MRI lesions at the initial clinical presentation. For that particular patient, she had normal MRI scan at first at her initial diagnosis of optic neuritis, and had MRI lesions on subsequent scans. The types of initial MRI lesions in 97 (94.2%) patients were presented in Table 2, including most frequently supratentorial (85, 82.5%), followed by infratentorial (28, 27.2%) and spinal cord (27, 26.2%) lesions, while the data were not available in the remaining 5 (4.85%) patients.

In 41 (39.8%) patients with evoked potential studies performed and with available results, at least one abnormal evoke potential study was found in 23 (22.3%) of them as shown in Table 2 including abnormal VEP (16, 15.5%), BAEP (6, 5.83%), and SSEP (10, 9.71%). Sixty-three (61.2%) and twenty-two (21.4%) patients were tested for serum antibodies against aquaporin-4 and myelin oligodendrocyte proteins, with all yielded negative result.

CSF was analysed with data available in 56 (54.3%) patients. Thirty-four (60.7%) of them were tested positive for OCBs. The mean CSF protein level was 0.37 g/L (SD 0.22) and CSF white blood cells count was 4.06 per µL (SD 6.49) with distribution shown in Figures 2 and 3 respectively.

Figure 2 A Box-and-whisker plot of CSF Protein level. The box represented the interquartile range (IQR), where the horizontal line within the box indicates the median. o: outlier greater than 1.5 IQR from the upper quartile boundary.

Figure 3 A Box-and-whisker plot of CSF WBC count per microlitre. The box represented the interquartile range (IQR), the horizontal line within the box indicated the median, and the whiskers extended to 1.5 IQR from the quartile boundaries. o: outlier between 1.5–3 IQR from the upper quartile boundary. *: outlier greater than 3 IQR from the upper quartile boundary.

Relapses, Progression and Death

In the 66 RRMS patients with clearly defined study period, 221 relapses were recorded over a mean study period of 16.5 (SD 10.5) years. The mean ARR was 0.25 (SD 0.287). Thirty-eight (36.9%) of the 83 patients with available EDSS data in EDSS had EDSS worsening. Six RRMS patients progressed from RRMS to SPMS.

Eight (7.77%) deaths were recorded during the study period, with a mean age of death of 56 (SD 10.3) years. Four of them died of pneumonia, whereas others died of septicaemia due to Klebsiella and Candida infections, toxic megacolon, and intracerebral haemorrhage, respectively. The cause of death of the remaining patient was uncertain and the case was referred to coroner. The result of the coroner report was not retrievable from the electronic patient record.

DMTs Use

One hundred thirty-seven DMT cycles were commenced in 76 (73.8%) patients. The most common DMT used was interferon – either interferon-beta 1a (Avonex®, Rebif®), or 1b (Betaferon®) (48, 35.0%), followed by dimethyl fumarate (DMF) (41, 30.0%), fingolimod (FTY) (14, 10.2%) and teriflunomide (TFN) (8, 5.84%). Other DMT agents (26 cycles, 19.0%) included traditional steroid-sparing agents like azathioprine, mycophenolate mofetil, methotrexate, and newer agents including cladribine, ocrelizumab, alemtuzumab, and rituximab.

In 76 (73.8%) patients with history of DMT use, a median of 2 DMTs were used. The mean duration between between diagnosis and first DMT treatment was 21.7 (SD 37.7) months. DMTs were not started in 27 (26.2%) patients, 13 ie almost half of them were due to patients’ reluctance towards treatment after physicians’ explanation. DMT was not indicated in 12 patients as there were no recent active attacks at the time of diagnosis, while the EDSS was above 6 in 1 patient that precluded DMT commencement. The record was not available for 1 remaining patient.

DMT Use Pattern

Amongst patients who have been put on DMTs, total number of DMT treatment cycles ranged from 1 to 5, with mean of 1.80 (SD 0.817). The vast majority of IFN (46 out of 48 cycles) was started in treatment-naïve patients without previous DMT used, whereas majority of DMF (27 out of 41 cycles), TFN (5 out of 8 cycles) and all FTY (14 out of 14 cycles) had one or more than one treatment cycles prior (Table 3). For patients having frequent DMT switch up to 5 treatment cycles, it was usually related to highly active disease course.

Baseline Characteristics with Respect to DMT Types

Among the 76 patients with history of DMT use, 56 were RRMS subtype. Baseline characteristics in each treatment subgroups (Table 4) included age of symptom onset, gender, smoking status, duration between symptom and diagnosis, types of symptoms and MRI lesions at initial presentation, CSF protein and WBC level, presence of OCB, abnormal evoked potentials, EDSS at diagnosis, and duration of DMT usage.

Effectiveness of DMTs

ARR with Respect to Each DMT Use

In 55 RRMS patients with 100 DMT treatment cycles, ARR was calculated for those with a clear definite study period. The ARR 2 years before and that after treatment were shown in Table 5. IFN (−0.492, p-value <0.001) and FTY (−0.557, p-value 0.038) were associated with statistically significant reductions in ARR. The use of DMF had a trend of ARR reduction (-0.095), though not statistically significant. On the other hand, ARR reduction (+0.465) was not shown in TFN subgroup. The number of cycles for DMTs other than IFN, DMF, TFN and FTY was too small for individual ARR analysis, or did not have statistically significant findings.

Persistence and Safety Profile of DMTs

Persistence and Discontinuation of DMTs

Out of the 76 patients with DMT use, a total number of 137 DMTs were used. Seventy-one (51.8%) treatments were continued throughout the study period in Table 6, most commonly DMF (34, 82.9%), followed by FTY (10, 71.4%). The median treatment duration ranges from 20.0 months in DMF to 58.5 months in IFN.

The Kaplan-Meier curve in Figure 4 portrayed the continuation of DMTs with respect to DMT types. No between-group statistical difference existed in the probability of DMT continuation with p-value of 0.367 in Log rank test.

Figure 4 Kaplan-Meier curves showing the probability of DMT continuation over time among patients with different DMT types. The x-axis represented the study period in months, where the y-axis shows the probability of treatment continuation. IFN (blue), DMF (red), TFN (green), FTY (Orange), and other DMTs (yellow) are plotted separately; censored data are denoted by crosses.

On the other hand, 48.2% of treatment discontinued, ie either stopped or switched to other DMTs, most seen in IFN (41, 29.9%), followed by TFN (4, 0.03%). Among them, 13 discontinued within first year, including 3 in TFN and 4 in IFN. The reasons of discontinuation included most due to side effects, intolerance, or patient preference (32, 23.3%), followed by disease activities (23, 16.8%), and other reasons included pregnancy and change of diagnosis.

Individual breakdown into DMT types listed in Table 6 with most common reason of discontinuation was side effects, intolerance in IFN (23 out of 41) and TFN (3 out of 4), and disease activities in DMF (7 out of 7).

Side Effects and/or Intolerance with Discontinuation of DMTs

Side effects, intolerance or personal preference led to discontinuation in 32 (23.4%) of the 137 treatment cycles. Table 7 summarised the side effects reported, though a large portion of the patients continued with the DMTs even with the side effects.

Fifteen (10.9%) IFN cycles were discontinued due to various side effects, most commonly due to injection reactions in 12 patients, and other reasons included mood disturbance, deranged liver function, and poor appetite. Four (2.92%) TFN cycles were discontinued due to intolerance and impaired liver function. Two (1.46%) FTY cycles were discontinued as potential culprit for impaired liver function and lymphopenia. Two (1.46%) patients discontinued azathioprine due to personal preference and proneness to infections. One patient switched to rituximab therapy, but she developed septicaemia afterwards, and then the patient refused subsequent cycles. No patients discontinued DMF due to side effects or intolerance.

The following Kaplan-Meier curve in Figure 5 portrayed the discontinuation of DMTs due to side effects and/or intolerance. There exists statistical significance between various DMTs with p-value of 0.013 by Log rank test. Performing the Cox regression, it was found that TFN was associated with an increased HR of 7.50 (Confidence interval (CI): 1.23–45.7), with statistical significance of p-value 0.029.

Figure 5 Kaplan-Meier curves showing the probability of DMT discontinuation over time among patients with different DMT types. The x-axis represented the study period in months, and the y-axis showed the probability of discontinuation for each therapy. Lines indicated specific therapies: IFN (blue), DMF (red), TFN (green), FTY (orange), and other DMTs (yellow). Censored data points are indicated with crosses. Discontinuation due to intolerance or side effects was lowest for DMF, with statistical significance is indicated (p = 0.013). * p-value less than 0.05 indicated statistical significance.

Disease Activities Associated with Discontinuation of DMTs

The following Kaplan-Meier curve in Figure 6 portrayed the discontinuation of DMTs due to disease activities with respect to DMT types. Twenty-three (16.8%) of the treatment cycles were terminated due to increased activities, including 11 (8.03%) of interferons, 7 (5.11%) of dimethyl fumarate, 2 (1.46%) of azathioprine, 1 (0.73%) of teriflunomide, fingolimod and cladribine respectively. No statistically significant inter-group difference was demonstrated by log rank test with p-value of 0.348.

Figure 6 Kaplan-Meier curves showing the probability of disease-modifying therapy (DMT) discontinuation due to disease activity over time, stratified by therapy type. The x-axis shows study period in months, and the y-axis displays probability of discontinuation. IFN (blue), DMF (red), TFN (green), FTY (Orange), and other DMTs (yellow) are plotted separately; censored data are denoted by crosses. The difference in rates of discontinuation across DMTs indicated no significant differences between therapies (p = 0.348).

In IFN groups, 7 of them were switched to fingolimod, while 4 of them switched to DMF. In DMF groups, all except 2 were switched to FTY whereas the remaining were switched to cladribine and alemtuzumab respectively. For FTY group, one switched to TFN, while another switched to alemtuzumab. Two of azathioprine therapies were switched to mycophenolate mofetil and methotrexate respectively. One patient switched use of cladribine to rituximab.

Other Reasons Associated with Discontinuation of DMTs

Two patients with the use of interferon therapies switched to azathioprine after progression from RRMS to SPMS. Four patients have once discontinued use due to pregnancy.

Azathioprine was commenced in 2 of the patients with initial diagnosis of NMOSD. It was subsequently switched to IFN and TFN, respectively, when the diagnosis of RRMS was established.

Prognostic Factors

Prognostic Factors in Disability and Disease Progression

Various demographic and clinical variables were compared (Table 8), in an attempt to identify their association with EDSS worsening in 83 patients.

Table 1 Demographic and Clinical Parameters for Analysis

Table 2 Clinical Characteristics of MS Patients

Table 3 Number of Prior DMTs Used on with Respect to DMT Types

Table 4 Baseline Characteristics of RRMS Patients with Respect to DMT Types

Table 5 ARR Prior to and After Treatment with Respect to DMT Types

Table 6 Continuation and Discontinuation of DMTs with Respect to DMT Types

Table 7 Side Effects Reported in Various DMTs

Table 8 Prognostic Factors with Respect to EDSS Worsening

Table 9 Prognostic Factors with Respect to ARR

It was found that EDSS more than or equal to 4.0 at diagnosis (OR: 4.56, p-value 0.0447), brainstem lesions on initial MRI scan (OR: 3.15, p-value: 0.039) were positively associated with worsening in EDSS. The presence of visual symptoms (OR: 0.144, p-value 0.007) were associated negatively associated with worsening in EDSS. These were associated with statistical significance. Upon binary logistic regression analysis, none demonstrated statistical significance.

Age of onset (OR 2.26, p-value 0.230), ever-smoker status (OR 2.57, p-value 0.145), progressive MS (OR 2.73, p-value 0.132), pyramidal (OR: 1.73, p-value 0.322) and sensory (OR: 2.17, p-value 0.114) symptoms, cerebellar (OR 2.09, p-value 0.193), and spinal cord (OR: 2.03, p-value 0.157) lesions at initial presentation, all had trend of higher odds ratio, though these were statistically insignificant.

Prognostic Factors in ARR

Table 9 listed various demographic and clinical variables, where their respective means of ARR were compared. The result showed that ever-smoker status (0.179, p-value 0.037), more than 10 white blood cells per µL in CSF (0.526, p-value 0.003) and first relapse within 6 months (0.262, p-value 0.006) were associated with higher ARR with statistical significance. OCB in CSF was, however, associated with lower ARR (0.198, p-value 0.043). Multivariate analysis was performed with linear regression, yet none demonstrated statistical significance.

Discussion

This local cohort study investigated the demographic and clinical characteristics of MS patients in Hong Kong and their DMT selection and preferences.

Patients Characteristics

The baseline characteristics were comparable to those of other Caucasian cohort studies,²⁰ which contrasted with a previous report that older age of onset, female preponderance was found in the Asian population.²¹ It is postulated that the previous cohort might include patients with NMOSD, and a lower availability of aquaporin-4 testing had back then given a limited understanding.

DMT Use

Effectiveness of DMT

The effectiveness of various DMTs was proven in various randomised controlled trials, as compared to placebo,^11,22–30 with some of them also compared to one another.^31–33 Head-to-head comparisons between different DMTs were not widely studied in the literature. In Hong Kong, where majority of MS patients were managed in Hospital Authority. We started from first line therapies like IFN, DMF and then escalated up to more highly efficacious therapy with disease activities.³⁴ Comparison was hence difficult due to this usual DMT treatment sequence.

In our study, significant ARR reductions were observed after DMT commencement of IFN, FTY, and other treatments, but not DMF and TFN. The reason could be due to small sample size, and the lateral switch of the DMTs. It was observed that a few of our patients switched from IFN to DMF and TFN after they were made available in our formulary, instead of after having disease activity as they were of good disease control all along. It might partly explain why there was no significant ARR reduction in our study.

Persistence and Discontinuation of DMT

In reality, the effectiveness is often limited by the non-compliance. Adherence to DMT has been shown to decrease risk of relapses.³⁵ Therefore, study towards the persistence of DMT is important. In our cohort, it was found that the overall discontinuation rate was 48.2%, and the discontinuation rates were similar in various subgroups except TFN, which was mostly in align with findings in a meta-analysis including real-world observational studies in the Western.³⁶ On the other hand, 9.49% discontinuation rate was observed in the first year, lower than the mean first-year discontinuation rates reported in the same meta-analysis.³⁶ As limited data in Asia was available, it is uncertain whether these data represent that Asian patients in general have a better tolerance towards DMT.

Side effects were one major factor for the discontinuation.^37,38 For IFNs, the need for self-injection precluded the persistence. Side effects such as injection reactions, flu-like symptoms in our study, were also commonly reported in previous literature. Despite so, most patients tolerated these side effects and continued their uses until the availability of oral alternatives available.

Common side effects of DMF, for instance, gastrointestinal upsets and flushing were reported in our study. However, these appeared well tolerated in our cohort and none discontinued due to side effects. In a multi-centre observational analysis involving mostly Caucasians,³⁹ DMF was recorded to have the highest discontinuation rate owing to side effects, whereas this is in contrast to an open-label extension study of DMF trial involving Japanese patients,⁴⁰ no discontinuation was observed with lower reduction in lymphocyte count and lower incidence of flushing.

In TFN subgroup of our cohort, elevated liver enzymes, gastrointestinal upset, hair loss were reported in 3 out of 8 treatment cycles, with 4 discontinuations observed. In other cohorts, for example, in a retrospective study using healthcare claim data in the United States and a subgroup of Asian patients in a TFN trial,^41,42 the discontinuation rate of TFN was similar compared with overall discontinuation rate. Hence, our observation of higher risk of deterioration with small size in the TFN subgroup required further studies with larger scale to support its significance.

In the studied population, it was observed that a portion of interferons were withheld during pregnancy with previous uncertainty of the safety of their use during pregnancy, either suggested by treating physician or patients themselves. As a result of a meta-analysis⁴³ showing similar rate in birth defects and emerging of post-market data of interferons, they are nowadays deemed safe to be continued according to the consensus published by the Association of British Neurologists,⁴⁴ and the category C warning was removed in the FDA in 2020.⁴⁴ In our cohort, none reported adverse pregnancy outcomes, though the size was too small to allow conclusions to be drawn.

Prognostic Factors

A number of environmental, demographic, clinical factors and biomarkers were studied in an attempt to spot out ones with prognostic values. Table 10 depicted factors that were associated with better and worse prognosis in MS found in the literature.

Table 10 Factors with Prognostic Values in MS Reported in Literature

Use of Outcome Measures

ARR and EDSS worsening which we used in our study were common markers to indicate disease activities and progression, respectively.^59–61 For relapse-related outcome measures, ARR is commonly used to reflect inflammatory activities as the data are readily accessible, at the expense of lacking specificity to severity of each attack.⁶¹ For measuring disability progression, EDSS worsening is also commonly used owing to its readiness, though it is limited by its insensitivity to functional system related and cognition-related deficit.⁶¹

In our cohort, we observed eight deaths during the study period, its small sample size however precluded further statistical analysis. Two patients had high EDSS score at the time of death with pneumonia as cause of death. It could be postulated to be related to immobilisation, or aspiration due to advanced neurological disease. Another 2 patients on azathioprine, an immunosuppressant, died of pneumonia and septicaemia. It was observed in a Canadian cohort that deaths were commonly caused by infective conditions.⁶² Identification of the prognostic factors to all these adverse outcomes could allow better understanding to this disease.

Environmental and Demographic Factors

In our study, we were able to identify ever-smoking status at diagnosis was associated with higher risk of EDSS worsening, consistent with result in a meta-analysis where ever-smoking status was associated with poorer outcome.⁶³ Male sex and age greater than 40, where some were reported to be associated with worse outcome with conflicting results,^{45–48,50,52,64} were not found to have worse outcomes in this study.

Clinical Factors

Optic neuritis at diagnosis was associated with a lower risk of attaining EDSS worsening. It was supported by some cohort studies, including one in Hong Kong.^1,52,65 Pyramidal and cerebellar symptoms were reported to have association with poor prognosis^50,52 though these were not demonstrated in our study.

We found that early relapse within 6 months was associated with a higher risk of ARR. Worse outcomes were found in several studies with a shorter time to reach a higher EDSS score.^52,66 We also noted a higher risk of EDSS worsening in patients with a higher baseline EDSS score, which concurred with a study where patients with EDSS greater than 2.0 were predictive of poor prognosis.⁶⁷

Biomarkers and Imaging Characteristics

We found that ARR was higher in the group with CSF pleocytosis. It was similarly observed in a cohort that a higher number of white blood cells in CSF, with cutoff of 10 cells, was associated with higher ARR in patients with RRMS.⁵⁴ Thus, CSF pleocytosis reflects a potential role as a surrogate marker to reflect disease progress in MS.

In our study, OCB was negatively associated with ARR and no effect on EDSS worsening. Despite its potentially useful role as a biomarker in predicting conversion of CIS to RRMS,⁶⁸ and a higher probability of worse outcome,⁴⁵ an observational cohort in Austria did not identify any relationship between the two.⁶⁴ Therefore, the role in predicting prognosis in RRMS was uncertain where future studies are warranted to confirm this association.

Brainstem, cerebellar and spinal cord lesions were found to have an association with a higher probability of EDSS worsening, though only brainstem lesions were found with statistical significance. It was also similarly found that baseline infratentorial lesions were strong predictors of progression from both CIS to RRMS, and RRMS to SPMS.⁵⁶

Some other biomarkers and MRI characteristics were implicated to have prognostic values.⁵¹ One of them is neurofilament light-chain protein,⁵⁵ a neuronal protein that is postulated to be released during axonal damage. Others included a number of MRI lesions, brain volume change and atrophy.⁵¹ Nevertheless, neurofilament testing was not available in Hospital Authority, and the MRI characteristics were not routinely reported by our local radiologists in general. Consequently, these markers were not included in our analysis.

Strength and Weakness

This study provided real-world experience in Hong Kong, regarding the effectiveness, persistence, and discontinuation of DMTs in the epoch with widely available oral options. Baseline characteristics of this local cohort were analysed so that several prognostic factors were found, which potentially aid clinicians in decision-making process.

Yet, there existed quite a few shortcomings, which included retrospective design, missing data, small sample size, and possible selection bias inherent in non-randomised treatment allocation.

A retrospective observational study design without control subgroups was subjected to a number of confounding factors such as disease severity, co-morbidity in the interpretation of efficacy and side effects. For instance, treatment allocation was determined by the attending neurologist’s clinical judgment rather than random assignment, confounding by indication cannot be excluded. Also, for those with better clinical course with relatively less relapses or even achieved remission, they were more likely to default follow-up.

Secondly, as a single centre study, its small sample size such that it lacked sufficient power for conducting multivariate analysis. Nevertheless, owing to relatively low prevalence, it encompassed quite a number of MS patients of Hong Kong already. Another important factor is the heterogeneity of our local MS patients, which rendered our review particularly difficult. Still, despite all these, we hoped to gain insights into the usage of DMTs in our local MS patients.

Thirdly, data collection was based on a review of both electronic and handwritten medical records. Variations in documentation completeness and interpretation could have introduced information bias, despite efforts to ensure accuracy through cross-checking.

Finally, missing data were present for certain variables, including EDSS scores and MRI data. Even though these cases were excluded from specific analyses, this might under- or over-estimate the true associations. The definite time of EDSS score change could not be determined with certainty, as it was not included as an outcome measurement in our cohort.

Conclusion

Multiple sclerosis is a chronic neurological illness that commonly affected the young individuals, which could pose significant disability and negative consequences to daily life.

In our study, the use of DMTs pattern appeared similar with other observational cohorts. IFN, FTY and other DMTs were associated with a significant ARR reduction, though the apparent lack of benefit in DMF, TFN could be due to inadequate power with the heterogeneous population. Similar persistence to DMTs with a significantly higher portion of TFN discontinued due to intolerance was demonstrated, though the finding is underpowered.

We identified several poor prognostic factors in our MS patients – ever-smoking status, EDSS more than or equal to 4, short period of relapse, CSF leucocytosis and brainstem MRI lesions. The good prognostic factors were the presence of visual symptoms and OCB in CSF. With the identification of the potential prognostic factors in this cohort, clinicians could make use of these to facilitate communication to allow patients to better understand their disease prognosis. Although no significant association with other factors could be found due to limited sample size, future studies with larger sample size, collaboration with multiple centres, and multivariate analysis were recommended to reduce influence of the other unmeasured factors and to confirm the association.

Overall, this study illustrated the beauty, and complexity in managing MS patients with all the considerations for patients’ clinical characteristics, diseases activity, risk appetite and treatment side effects so as to reach an informed decision on the use of DMTs in our local MS patients.

Disclosure

The authors report no conflicts of interest in this work.

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About multiple myeloma

Multiple myeloma is the third most common blood cancer globally and is generally considered treatable but not curable.^1,2 There are approximately 180,000 new cases of multiple myeloma diagnosed globally each year.³ Research into new therapies is needed as multiple myeloma commonly becomes refractory to available treatments.⁴ Many patients with multiple myeloma, including approximately 70% in the US, are treated in a community cancer setting, leaving an urgent need for new, effective therapies with manageable side effects that can be administered outside of an academic centre.^5,6,7

About myelofibrosis

Myelofibrosis is a rare blood cancer that disrupts the body’s normal production of blood cells because of dysregulated JAK-signal transducer and activator of transcription protein signalling. The clinical hallmarks of myelofibrosis are splenomegaly (enlarged spleen), severely low blood counts, including anaemia and thrombocytopenia, and debilitating constitutional symptoms, such as fatigue, night sweats and bone pain, attributable to ineffective haematopoiesis and excessive production of proinflammatory cytokines.^8,9

About belantamab mafodotin

Belantamab mafodotin is a monoclonal ADC (antibody-drug conjugate) comprising a humanised BCMA (B-cell maturation antigen) conjugated to the cytotoxic agent monomethyl auristatin F via a non-cleavable linker. The drug linker technology is licensed from Seagen Inc.; the monoclonal antibody is produced using POTELLIGENT Technology licensed from BioWa Inc., a member of the Kyowa Kirin Group.

In October 2025, the US FDA approved¹⁰ belantamab mafodotin under the brand name Blenrep in combination with bortezomib and dexamethasone (BVd) for the treatment of adult patients with relapsed or refractory multiple myeloma who have received at least two prior lines of therapy, including a proteasome inhibitor and an immunomodulatory agent. 

Belantamab mafodotin  in combination with bortezomib and dexamethasone and belantamab mafodotin in combination with pomalidomide and dexamethasone are approved in 2L+ relapsed or refractory multiple myeloma in the European Union¹¹, UK¹², Japan¹³, Canada, Switzerland and Brazil. 

Applications are currently under review in other markets globally, including China¹⁴ where the application is based on the results of DREAMM-7 and has been granted Breakthrough Therapy Designation and Priority Review.  

Indication

In the US, Blenrep is indicated in combination with bortezomib and dexamethasone (BVd) for the treatment of adult patients with relapsed or refractory multiple myeloma who have received at least two prior lines of therapy, including a proteasome inhibitor and an immunomodulatory agent.

Please see accompanying US Prescribing Information.

About momelotinib

Momelotinib has a differentiated mechanism of action, with inhibitory ability along three key signalling pathways: Janus kinase (JAK) 1, JAK2, and activin A receptor, type I (ACVR1).^15,16,17,18 Inhibition of JAK1 and JAK2 may improve constitutional symptoms and splenomegaly.^15,16,17 Additionally, inhibition of ACVR1 leads to a decrease in circulating hepcidin levels, potentially contributing to anaemia-related benefit.^15,16,17,18

In September 2023, the US Food and Drug Administration approved¹⁹ momelotinib under the brand name Ojjaara for the treatment of intermediate or high-risk myelofibrosis, including primary myelofibrosis or secondary myelofibrosis (post-polycythaemia vera and post-essential thrombocythemia), in adults with anaemia.

In January 2024, the European Commission granted marketing authorisation²⁰ for momelotinib for disease-related splenomegaly (enlarged spleen) or symptoms in adult patients with moderate to severe anaemia who have primary myelofibrosis, post polycythaemia vera myelofibrosis or post essential thrombocythemia myelofibrosis and who are Janus kinase (JAK) inhibitor naïve or have been treated with ruxolitinib. Momelotinib was also approved²¹ by the Medicines and Healthcare products Regulatory Agency (MHRA) in the United Kingdom to treat the symptoms experienced by adult myelofibrosis patients who have moderate or severe anaemia.

In June 2024, the Japan Ministry of Health, Labour and Welfare (MHLW) approved²² momelotinib for the treatment of myelofibrosis. Momelotinib is currently approved in 21 countries and applications are under review in other markets globally. 

Important information for momelotinib in the EU

Indication

Momelotinib is indicated for the treatment of disease-related splenomegaly (enlarged spleen) or symptoms in adult patients with moderate to severe anaemia who have primary myelofibrosis, post polycythaemia vera myelofibrosis or post essential thrombocythaemia myelofibrosis and who are Janus kinase (JAK) inhibitor naïve or have been treated with ruxolitinib.

Refer to the Omjjara EMA Reference Information for a full list of adverse events and the complete important safety information in the EU.

GSK in oncology

Our ambition in oncology is to help increase overall quality of life, maximise survival and change the course of disease, expanding from our current focus on blood and women’s cancers into lung and gastrointestinal cancers, as well as other solid tumours. This includes accelerating priority programmes such as antibody-drug conjugates targeting B7-H3 and B7-H4, and IDRX-42, a highly selective KIT tyrosine kinase inhibitor.

About GSK

GSK is a global biopharma company with a purpose to unite science, technology, and talent to get ahead of disease together. Find out more at www.gsk.com.

Cautionary statement regarding forward-looking statements

GSK cautions investors that any forward-looking statements or projections made by GSK, including those made in this announcement, are subject to risks and uncertainties that may cause actual results to differ materially from those projected. Such factors include, but are not limited to, those described in the “Risk Factors” section in GSK’s Annual Report on Form 20-F for 2024, and GSK’s Q3 Results for 2025.

AMSTERDAM – Stellantis CEO Antonio Filosa will participate in a fireside chat on Thursday, December 4, 2025, from 3:05 p.m. to 3:40 p.m. CET / 9:05 a.m. to 9:40 a.m. EST at Goldman Sachs 17^th Annual Industrials & Autos Week.

To watch the live session, visit the following webcast link: https://kvgo.com/gs/stellantis-december-2025

Details for watching the fireside chat are also available under the Investors section of the Stellantis corporate website. For those unable to attend the live session, a recorded replay will be accessible following the event.

 

 

About Stellantis

Stellantis N.V. (NYSE: STLA / Euronext Milan: STLAM / Euronext Paris: STLAP) is a leading global automaker, dedicated to giving its customers the freedom to choose the way they move, embracing the latest technologies and creating value for all its stakeholders. Its unique portfolio of iconic and innovative brands includes Abarth, Alfa Romeo, Chrysler, Citroën, Dodge, DS Automobiles, FIAT, Jeep_®, Lancia, Maserati, Opel, Peugeot, Ram, Vauxhall, Free2move and Leasys. For more information, visit www.stellantis.com.

 

 

(more…)

LUXEMBOURG, Dec. 1, 2025 /PRNewswire/ — Ardagh Metal Packaging S.A. will participate in Citi’s 2025 Basic Materials Conference on Wednesday, December 3, 2025.

Oliver Graham, Chief Executive Officer, will participate in a fireside chat on Wednesday, December 3, 2025 at 13:40 US Eastern Time. A live webcast of the fireside chat will be available on the Company’s investor relations website and under the following Link.

Following the conclusion of the fireside chat, a replay of the event will be made accessible later that day using the same link.

To view this release online and get more information about Ardagh Metal Packaging please visit: https://www.ardaghmetalpackaging.com/investors

About Ardagh Metal Packaging
Ardagh Metal Packaging (AMP) is a leading global supplier of sustainable and infinitely recyclable metal beverage cans to brand owners globally. An operating business of sustainable packaging business Ardagh Group, AMP is a leading industry player across Europe and the Americas with innovative production capabilities. AMP operates 23 metal beverage can production facilities in nine countries, employing more than 6,000 people with sales of approximately $4.9 billion in 2024.

Contacts:

Investors:
Email: [email protected]

SOURCE Ardagh Metal Packaging S.A.

Directive gives companies 90 days to ensure Sanchar Saathi app is pre-installed on new mobile phones.

The Indian government has instructed smartphone makers to pre-install a state-owned cybersecurity app on all new devices in a bid to tackle online scams and other crimes, according to reports.

The November 28 order – the existence of which was reported by Reuters news agency and Indian media on Monday, three days after it was privately sent to manufacturers – gives the companies 90 days to ensure that the app, Sanchar Saathi or “communication companion”, is pre-installed on new mobile phones, with a requirement that users cannot disable it.

The directive of the Department of Telecommunications (DoT) also requires phone companies to push a software update for installing the app on devices already in circulation, the reports said.

The app, which was released in January, is currently available for download, with India’s 1.2 billion smartphone users having the option to install it.

The government says the app is essential to combat “serious endangerment” of cybersecurity from duplicate or spoofed International Mobile Equipment Identity (IMEI) numbers – the code assigned to each device that is used to cut off network access for phones that are reported stolen.

According to government figures, users have downloaded the app more than five million times since its launch, helping to block more than 3.7 million stolen or lost mobile phones and blocking more than 30 million fraudulent connections, Reuters reported.

In that time, the app has helped recover more than 700,000 lost phones, according to the figures.

Apple ‘likely to resist’

But the order is likely to face pushback from US tech giant Apple, which has previously clashed with India’s telecoms regulator over a government antispam mobile app, as well as privacy advocates, Reuters reported.

Apple has internal guidelines against installing any third-party apps – including government-developed ones – prior to the sale of a device, a source with direct knowledge of the matter told the news agency.

Tarun Pathak, a research director at technology market research firm Counterpoint, told Reuters that Apple had previously refused similar requests from governments.

“It’s likely to seek a middle ground: instead of a mandatory pre-install, they might negotiate and ask for an option to nudge users towards installing the app,” Pathak said.

Mishi Choudhary, a lawyer who works on internet advocacy issues, told the agency that the order was concerning, as it “effectively removes user consent as a meaningful choice”.

There was no immediate comment about the reports by the DoT.

The directive follows similar moves by governments, most recently Russia, to crack down on the use of phones for fraud and push state-backed apps.

Introduction

Shaoyao Gancao Decoction (SGD) is a famous traditional Chinese medicine (TCM) formula originating from the Treatise on Febrile diseases written by Zhongjing Zhang during the Han Dynasty.^1,2 It is composed of Paeoniae Radix Alba (PRA, called Baishao in China) and Glycyrrhizae Radix et Rhizoma (GRR, called Gancao in China) at a ratio of 1:1, and is widely used in the treatment of digestive system diseases and gynecological disorders.^2,3 Recent studies have demonstrated that SGD possesses multiple pharmacological effects, including spasmolysis, analgesia, and anti-inflammatory effects, and has various chemical components, including paeoniflorin (Pae), oxypaeoniflorin, albiflorin, benzoylpaeoniflorin, glycyrrhizic acid (GL), liquiritin apioside, liquiritin, liquiritigenin.^1–4 However, these components are predominantly glycosides or flavonoids, which exhibit poor oral bioavailability because of their high polarity or low solubility. In particular, Pae, a P-glycoprotein (P-gp) substrate, was demonstrated to be extremely poor absorption with absolute oral bioavailability ranging from 3% to 4%.^5,6

Notably, TCM herbal formulas exhibit an intriguing characteristic that certain phytochemicals with inherently low bioavailability show dramatically increased absorption when orally administered with TCM formula decoctions. Representatively, the bioavailability of Pae was significantly improved by the oral administration of SGD,⁷ but the mechanism underlying the bioavailability improvement remains unclear. There have been some studies on the potential mechanisms by which active ingredients of licorice affect Pae absorption. However, there are contradictory findings regarding the effects of licorice constituents on P-gp. Glycyrrhetinic acid (GA) and 18β-GA (a predominant conformation of GA) have shown P-gp inhibitory activity in certain studies.^8,9 In contrast, other studies have reported that GA activated the function and expression of P-gp.^10,11 Additionally, GL has been reported to decrease the absorption of Pae by inducing the activity of P-gp.¹² In another study, GL exhibited concentration-dependent effects on intestinal Pae absorption: 1 mM GL significantly increased absorption in the jejunum after 45 min and in the ileum after 90 min, whereas 100 μM GL inhibited absorption in the jejunum.¹³ Therefore, the underlying mechanisms by which oral administration of SGD improves bioavailability of Pae remain to be elucidated due to these inconsistent findings.

Recently, self-assemblies of TCM, also known as supramolecules and natural nanoparticles, have received growing attention as they can not only help elaborate the scientific connotation of TCM compatibility, but also be used as drug carriers to improve the bioavailability and biological activities of medicinal components.^14–17 Self-assemblies in TCM decoctions, particularly in TCM formulas, result from high-temperature boiling-induced non-covalent interactions (hydrogen bonding, electrostatic forces, ionic interactions, and hydrophobic effects) among the active components.^15,16 Our previous study has demonstrated that GL can self-assemble into nanomicelles, significantly enhancing the intestinal absorption and bioavailability of Pae.¹⁸ Glycyrrhiza protein (GP) has also been reported to self-assemble into functional nanoparticles that are capable of significantly increasing solubility, promoting absorption, and enhancing the bioactivity of drugs.^19–22 The self-assembled GP nanoparticles were also found to increase the intestinal absorption of Pae.²³ However, the formation of self-assembled nanoparticles in SGD (SGD-SAN) is not achieved solely by GL or GP, but rather results from the synergistic interaction of multiple components. It remains unclear whether the nano-assemblies in SGD differ fundamentally from either GL self-assembled nanomicelles (GL-SNM) or GP self-assembled nanoparticles (GP-SAN).

Therefore, the present study was performed to systematically compare the in vitro and in vivo characteristics of three nano-assemblies of different components from SGD (SGD-SAN, GL-SNM, and GP-SAN), with particular emphasis on their differential effects on oral absorption of Pae. SGD-SAN was separated and then identified for its composition. GL-SNM and GP-SAN were self-assembled according to the main components obtained from the composition analysis of SGD-SAN and loaded with Pae, named Pae GL-SNM and Pae GP-SAN, respectively. The SGD-SAN, Pae GL-SNM, and Pae GP-SAN were characterized in terms of particle size, zeta potential, morphology, drug loading, and in vitro release behavior. The single-pass intestinal perfusion and pharmacokinetic studies of SGD-SAN, Pae GL-SNM, and Pae GP-SAN following oral administration were performed to evaluate their absorption-enhancing effect. These comprehensive comparisons will elucidate the structure-function relationships underlying SGD’s enhancement of Pae absorption, which is critical for revealing the synergistic mechanisms of multi-component interactions in TCM and clarifying the scientific basis of TCM compatibility. Moreover, clarifying these nano-assemblies’ contributions may guide the development of novel TCM-based drug delivery systems and more effective TCM-derived formulations, providing support for the modern application of TCM.

Materials and Methods

Materials

PRA and GRR were purchased from Jiangxi Peng’s Gaoyaotang Sliced Herb Co., Ltd. The herbal materials were authenticated by the Chief Pharmacist Ping Ying from the Department of Pharmacy, Jiangxi Provincial People’s Hospital (the First Affiliated Hospital of Nanchang Medical College), PR China. Pae (purity > 95%), GL (purity > 95%), Pae reference standard (purity > 98%), and puerarin (internal standard, purity > 98%) were obtained from Baoji Herbest Bio-Tech Co., Ltd (Xian, PR China). HPLC-grade methanol and acetonitrile were purchased from Sigma-Aldrich (USA). 3500 Da dialysis bag was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade.

Animals

Male Sprague-Dawley rats (weight 180–200 g, 6 weeks old) were obtained from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). All rats were housed in an environmentally controlled breeding room (25 ± 2 °C, 60% humidity, and 12 h cycle of light and dark) and received ad libitum access to water and food. All animal experiments were conducted in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of Nanchang Medical College (No. NYLLSC20240408).

Separation and Composition Analysis of SGD-SAN

Separation of SGD-SAN

SGD-SAN was separated as follows:²⁴ PRA and GRR herbs were weighed at a ratio of 1:1 (25 g + 25 g) and soaked in deionized water (500 mL) for 30 min. The mixture was boiled twice with 10- and 8-fold volumes (v/w) of deionized water (500 mL and 400 mL per 50 g herbs), each for 1 h. After filtration through eight layers of gauze, the two decoction liquids were combined and concentrated to 1 g/mL (crude drug equivalent) by rotary evaporation at 50 °C under reduced pressure (−0.08 MPa). After SGD preparation, SGD-SAN was obtained using a combined centrifugation-dialysis approach. SGD (50 mL) was centrifuged at 4000 rpm for 30 min to remove the precipitates. The supernatant (5 mL) was dialyzed against 200 mL of deionized water for 30 min using a 3500 Da molecular weight cutoff (MWCO) dialysis bag (25 mm flat width) at room temperature with constant agitation at 200 rpm, and then the liquids in dialysis bag was centrifuged at 13000 rpm for 30 min to remove the precipitates. The dialysis and centrifugation operations were repeated twice. Through the above operations, the liquid inside the dialysis bag was freeze-dried and collected as SGD-SAN.

Composition Analysis of SGD-SAN

The composition of SGD-SAN was analyzed using a BCA assay kit, phenol-sulfuric acid method, and high-performance liquid chromatography (HPLC). The protein content of SGD-SAN was determined using a BCA assay kit according to the manufacturer’s instructions. The polysaccharide content of the SGD-SAN was measured by the phenol-sulfuric acid method.²⁵ About of 20 mg SGD-SAN was dispersed in 10 mL of methanol, followed by ultrasonication for 10 min. After centrifugation at 8000 rpm for 10 min, the supernatant was filtered through 50 nm filters (Sigma-Aldrich, St Louis, MO, USA) for quantitative analysis of the main components by HPLC using an LC-20AD HPLC system (Shimadzu, Tokyo, Japan). Chromatographic separation was performed on an Inertsil^® ODS-3 column (250 mm × 4.6 mm, 5 µm, Shimadzu, Tokyo, Japan) using a mobile phase of acetonitrile and 0.1% phosphoric acid solution with gradient elution (Table 1). The flow rate was maintained at 1.0 mL/min, with the column temperature set at 30 °C. Detection was performed at a wavelength of 237 nm and the injection volume was 10 μL.

Table 1 Gradient Elution Conditions for Mobile Phase

Extraction of GP and Preparation of Pae GP-SAN

GP extraction from GRP was performed by the acetone precipitation method²² with some modifications. Briefly, GRP decoction pieces were mixed with deionized water at ratio of 1:5 (w/v) and extracted under ultrasonication for 30 min at 60 °C. The resulting mixture was filtered through a double-layer filter cloth and centrifuged at 4,000 rpm for 15 min to remove residues. The supernatant was precipitated with 1.5 times volume of acetone, incubated at −20 °C for 30 min. The precipitates were collected by centrifugation at 4,000 rpm for 15 min and then freeze-dried to yield GP. The obtained GP was determined by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were mixed with loading buffer and then heated at 95 °C for 10 min for protein denaturation. The denatured protein samples were separated by SDS-PAGE at 80 V to 120 V, and the protein bands were stained with Coomassie Brilliant Blue and compared with the protein standard.

The Pae GP-SAN was prepared by an ultrasonic dispersion method. Briefly, 16 mg of GP and 12 mg of Pae were added to 4 mL of deionized water and sonicated (JK-50B, Hefei Jinnick Medical Technology Co., Ltd., Hefei, China) at 250 W (50 Hz) for 25 min. After cooling to room temperature, the mixture was filtered through a 0.80 µm filter to obtain Pae GP-SAN.

Preparation of Pae GL-SNM

The Pae GL-SNM was fabricated via the film dispersion method, with a formulation consistent with that of our previous study.¹⁸ Briefly, GL (40 mg) was dissolved in 10 mL of absolute ethanol in a 50 mL pear-shaped flask and then evaporated by rotary evaporator (RE-2000B, Shanghai Yarong Biochemistry Instrument Factory, Shanghai, China) at 50 °C to obtain a thin film of GL. The obtained film was hydrated with 9.2 mL of deionized water through ultrasonication in a 50 °C water bath to form a micellar suspension. Then, 10 mg of Pae was dissolved in 0.8 mL of ethanol and added dropwise to the micellar suspension at 50 °C under continuous magnetic stirring (MS-H280-Pro, Dragon Laboratory Instruments Ltd., Beijing, China) at 500 rpm. After cooling to room temperature, the resulting mixture was sonicated for 30 min in an ice-water bath using an ultrasonic cell disintegrator (JY92-IIN, Ningbo Scientz Biotechnology Co., Ltd., China) at 240 W with a 3 s pulse interval. The ethanol was removed by rotary evaporation, and the volume was adjusted to 10 mL with deionized water. Finally, Pae GL-SNM was obtained by filtration through 0.80 µm microporous membrane filters.

Characterization of Nano-Assemblies

Size Distribution and Zeta Potential Analysis

The particle size and polydispersity index (PDI) of SGD-SAN, Pae GP-SAN, and Pae GL-SNM were determined by dynamic light scattering (DLS) using a Zeta-sizer (Malvern Zetasizer Pro, Worcestershire, UK). Samples were appropriately diluted with deionized water, equilibrated at 25 °C for 2 min prior to measurement, and analyzed at 25 °C with a scattering angle of 173°. Zeta potential was estimated by determining the electrophoretic velocity of the particles using the same instrument.

Morphology Evaluation

The morphologies of SGD-SAN, Pae GP-SAN, and Pae GL-SNM were observed by transmission electron microscopy (TEM, Tecnai G2F20, FEI, Eindhoven, Netherlands). After appropriate dilution with deionized water, one drop of the sample was deposited onto a 200-mesh copper grid, blotted with filter paper, and dried at room temperature. Images were obtained by TEM at 80 kV for morphological evaluation.

Encapsulation Efficiency and Drug Loading

The drug encapsulation efficiency (EE) and drug loading (DL) of Pae GP-SAN and Pae GL-SNM were determined by ultrafiltration method.^18,26 Briefly, 0.4 mL of sample was placed into the upper chamber of ultrafiltration centrifugal tube tubes (3 kDa, Millipore Corporation, Billerica, MA, USA) and centrifuged at 8000 rpm for 10 min. The filtrate was diluted 10-fold with methanol and the free Pae concentration was quantified by HPLC (LC-20AD, Shimadzu, Tokyo, Japan). The chromatographic separation was carried out on Inertsil^® ODS-3 column (250 mm × 4.6 mm, 5 µm, Shimadzu, Tokyo, Japan) with column temperature maintained at 40 °C and mobile phase of acetonitrile and 0.1% phosphoric acid (17: 83, v/v) at a flow rate of 1 mL/min. Detection was performed at 231 nm with an automated injection volume of 20 μL. The total Pae content in Pae GL-SNM or Pae GP-SAN was measured after extraction with 50-fold methanol in an ultrasonic bath. The EE and DL were calculated as follows:

where W_t is the total amount of Pae in Pae GP-SAN or Pae GL-SNM, W_f is the amount of free Pae unencapsulated in Pae GP-SAN or Pae GL-SNM, W_c is the total amount of GL or GP added. The drug loading (DL) of SGD-SAN was calculated by the Pae content in SGD-SAN, whereas the drug encapsulation efficiency (EE) was calculated as the ratio of the Pae content in SGD-SAN to the total Pae in SGD.

In vitro Drug Release

The Pae release from SGD-SAN, Pae GP-SAN, and Pae GL-SNM in vitro was investigated using a dialysis method and compared with that of Pae solution. The simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8) were selected as release media, respectively. All samples with dose equivalent to 10 mg Pae were placed into a dialysis bag (MWCO: 3500 Da; flat width: 25 mm, effective length: 30 mm). The bag was mounted with its symmetrical axis perpendicular to the direction of horizontal stirring to minimize boundary layer effects. Both ends were sealed with clips and immersed into 200 mL of release medium maintained at 37 ± 1 °C, and stirred at 100 rpm. At various time points (0.25, 0.50, 0.75, 1, 2, 4, 6, 8, and 12 h), 1 mL aliquots were withdrawn and replaced with an equal volume of media. Each sample was filtered through a 50 nm filter, and the Pae concentration was determined by the same HPLC method as described in the determination of EE and DL. Drug release profiles were plotted as the cumulative % drug release versus time.

Single-Pass Intestinal Perfusion Study

The intestinal absorption of SGD-SAN, Pae GP-SAN, and Pae GL-SNM was investigated by in situ single-pass intestinal perfusion method^18,27,28 and compared with that of Pae solution. Male SD rats were fasted for 12h with free access to water before the perfusion experiment, and then were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). The anesthetized rats were fixed in a supine position on a homeothermic blanket to maintain normal body temperature, and a midline incision of the abdomen was made. Approximately 10 cm segment of either the jejunum or ileum was carefully cannulated on two ends with plastic tubing and ligated using silk sutures. The dissected intestinal segments were gently rinsed with 37 °C saline to clear the segment and then attached to a peristaltic pump (L100-1S-1/DG-2, Baoding Longer Precision Pump Co., Ltd., Baoding, China). The entire surgical area was covered with a piece of sterile absorbent gauze wetted with saline (37 ± 1°C). Initially, the intestinal segment was perfused with blank Krebs-Rings (K-R) solution at a flow rate of 0.2 mL/min for 30 min to remove any residue. Then, the K-R solution containing different Pae samples (SGD-SAN, Pae GL-SNM, Pae GP-SAN, and Pae solution) at the same Pae concentration of 20 µg/mL²⁹ were perfused at a flow rate of 0.2 mL/min for 30 min to ensure steady-state conditions, and the outlet perfused samples were collected in pre-weighed vials at 15 min intervals up to 90 min and then weighed. The perfusion rate of 0.2 mL/min is designed to simulate the physiological state, reduce intestinal injury, and is physiologically equivalent to the 2.0–3.0 mL/min rate used in human jejunal perfusion.^30–32 The collected samples were stored at −20 °C until analysis by the same HPLC method as described in determination of EE and DL. Finally, the animals were euthanized with an overdose of isoflurane, and the length and inner diameter of the intestinal segment were measured. The gravimetric method was used to determine net water flux (water absorption and efflux in the intestinal segment).^27–29 The absorption rate constant (K_a) and effective permeability coefficient (P_eff) of Pae in intestinal segments were calculated using the following equations:

where and  are the concentrations of the test drug in the effluent perfusate through the inlet and outlet tubes (μg/mL), respectively.  and  represent the inlet and outlet volumes of the effluent perfusate (mL), respectively. is the perfusion volume flow rate (mL/h). and are the radius and the length of the perfused intestinal segment, respectively.

Pharmacokinetics Study

All rats were acclimated for one week and fasted for 12 h before the experiments. Twenty-four rats were randomly divided into four groups of six animals per group and received Pae solution, SGD-SAN, Pae GL-SNM, and Pae GP-SAN via gavage at a dose of 100 mg/kg Pae.¹⁸ After administration, approximately 0.5 mL of whole blood was collected from the orbital venous plexus at 0.083, 0.167, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12 and 24 h, and then placed into heparinized tubes. The plasma samples were obtained by centrifugation at 5000 rpm for 10 min, and stored at −80 °C until analysis.

The plasma samples were processed according to our previous study,¹⁸ and the Pae concentrations in plasma was determined by a validated HPLC/MS/MS system with puerarin as an internal standard (IS). An LC-30AD HPLC system (Shimadzu, Tokyo, Japan) was used for chromatographic separation with a Shim-pack Scepter C18 column (2.1 × 100 mm, 3 µm particle size, Shimadzu, Tokyo, Japan) maintained at 40 °C. The mobile phases consisted of 0.1% aqueous formic acid (A) and methanol (B) with gradient washing as follows: 0 ~ 1.1 min, 5%B; 1.1 ~ 2.4 min, 5 ~ 95% B; 2.4 ~ 4.0 min, 95%B; 4.0 ~ 4.5 min, 95 ~ 5%B; 4.5 ~ 6.0 min, 5%B. The flow rate was 0.3 mL/min and the injection volume was 5 µL. A TRIPLE QUAD^TM 4500MD mass spectrometer (AB SCIEX, Massachusetts, USA) was interfaced via electrospray ionization (ESI) source operating in positive ion mode at 550°C. The nebulizer gas (Gas 1), heater gas (Gas 2), curtain gas (CUR), and collision activated dissociation gas (CAD) were set to 55, 55, 33, and 8 instrument units, respectively. The quantification of Pae and Pue (IS) were quantified in the MRM mode using the pseudo-molecular (Q1) to fragment (Q3) ion transitions and the optimal declustering potential (DP), collision energy (CE), collision cell exit potential (CXP), and entrance potential (EP), as shown in the following conditions (Table 2).

Table 2 Mass Spectrometry Conditions

Determination of Dominant Driving Forces in Self-Assemblies

To elucidate the dominant driving forces for the formation of SGD-SAN, Pae GP-SAN, and Pae GL-SNM, chemical interference agents (NaCl, urea, and Tween 20) were added, coupled with particle size detection, to identify the types of intermolecular forces in the self-assemblies.^20,33,34

Statistical Analysis

The main pharmacokinetic parameters were calculated by DAS 2.0. All the experimental data were expressed as the mean ± standard deviation (mean ± SD). The SPSS 21.0 software was used to analyze the statistical data. First, the normality test and homogeneity of variance test of the data were performed. If the data meet the assumptions of normal distribution and homogeneity of variance, one-way analysis of variance (ANOVA) followed by the least significant difference post-hoc test was used to compare the differences among these groups. Otherwise, it was determined by Games–Howell test (heterogeneous variance) or Mann–Whitney U-test (non-normal distribution). Probability values P < 0.05 meant the difference was statistically significant.

Results and Discussions

Separation and Composition Analysis of SGD-SAN

According to our separation method, the average yield of SGD-SAN is (12.93 ± 0.58) %. Nine active ingredients were determined by HPLC. As shown in Figure 1A, the contents of isoliquiritin, liquiritigenin, benzoylpaeoniforin, and isoliquiritigenin were less than 0.2%, the contents of alibiflorin, liquiritin apioside, and liquiritin were about 1.25%, 0.97%, and 1.18%, while the contents of Pae and GL were 4.54% and 4.32%, respectively. These results suggested that Pae and GL were the main active ingredients. The protein content of SGD-SAN determined by BCA kit was about 61.91% (Figure 1B), accounting for most of the weight of SGD-SAN. In addition, the polysaccharide content of SGD-SAN was about 37.58%, while total content of 9 kinds of active ingredients was 12.69% (Figure 1B). The total content of all ingredients exceeded 100% (112.18%), which may be attributed to the overestimation of protein quantification owing to the presence of glucose³⁵ and repeated measurement of part carbohydrate components due to the presence of glycosides.

Figure 1 Composition analysis of SGD-SAN. (A). Composition of SGD-SAN with standard substances by HPLC (Data are presented mean ± SD.). (B). Proportion of proteins, polysaccharide, and active ingredients in SGD-SAN (Data are presented mean ± SD.). (C). SDS-PAGE bands of GP.

Preparation of Pae GP-SAN and Pae GL-SNM

To clarify the effects of nano-assemblies in SGD on Pae bioavailability, two new nanoformulations were prepared based on the chemical composition of SGD-SAN and their self-assembly properties. In our previous study, nanoparticles were mainly observed in single decoction of GRR and co-decoction of GRR and PRA, but only few nanoparticles were formed in single decoction of PRA²⁴ We speculated that nano-assemblies in SGD were mainly formed from the GRR components. The protein accounting for most of the weight of SGD-SAN was found to mainly come from GRR,³⁶ and can self-assemble into nanostructures with functional capability to encapsulate active ingredients.^19–22 Therefore, GP was extracted by an acetone precipitation method²² with a modification and was exploited to fabricate Pae GP-SAN. The extracted GP was characterized by SDS-PAGE, and the band of GP was located between 25–35 kDa (Figure 1C). In addition, GL was the main active ingredient and has been reported to form nanomicelles by self-assembly to improve the oral absorption of natural molecules and therapeutic agents.^18,37–39 Therefore, Pae GL-SNM was fabricated via the film dispersion method, with a formulation consistent with that of our previous study.¹⁸

Characterization of Nano-Assemblies

The physicochemical properties of the three nano-assemblies are presented in Table 3 and Figure 2. Significant differences were observed in the physicochemical properties of the three nano-assemblies. The mean particle sizes of SGD-SAN, Pae GL-SNM, and Pae GP-SAN were around 130, 150 and 180 nm (Figure 2 A1-A3), respectively, and their PDI were 0.354, 0.167, and 0.154, respectively. The PDI value exceeding 0.3 for SGD-SAN reflects a broad particle size distribution, whereas both Pae GL-SNM and Pae GP-SAN exhibited PDI values below 0.2, suggesting a narrow size distribution.⁴⁰ SGD-SAN exhibited the minimum particle size and maximum PDI, which may be due to its complex composition and sources. Repeated dialysis and centrifugation effectively removed larger particles, resulting in reduced particle size. The complex composition of SGD-SAN may contribute to its instability, leading to a broad particle size distribution. The TEM images demonstrated that SGD-SAN, Pae GL-SNM, and Pae GP-SAN were spherical in shape (Figure 2 B1-B3), with estimated average sizes of 50–300 nm for SGD-SAN and 100–200 nm for Pae GL-SNM and Pae GP-SAN, which is similar with the DLS measurements.

Table 3 Physicochemical Properties of Nano-Assemblies (Mean ± SD, n=3)

Figure 2 Particle size distribution and SEM images of Pae nano-formulations. (A1) and (B1) representing particle size distribution (PS:133 ± 13 nm, PDI:0.354 ± 0.015) and SEM images of SGD-SAN; (A2) and (B2) are particle size distribution (PS:154 ± 8 nm, PDI:0.167 ± 0.013) and SEM images of Pae GL-SNM; (A3) and (B3) are particle size distribution (PS:184 ± 7 nm, PDI:0.154 ± 0.009) and SEM images of Pae GP-SAN.

Both SGD-SAN and Pae GP-SAN exhibited zeta potentials of approximately −15 mV, suggesting limited colloidal stability, whereas Pae GL-SNM demonstrated a substantially higher absolute zeta potential (−35.88 mV), which was associated with enhanced stability due to stronger electrostatic repulsion.¹⁸ The similar zeta potentials observed for SGD-SAN and Pae GP-SAN may be attributed to their shared protein constituents, which likely dominate the surface charge characteristics of these nanoparticles. The EE of SGD-SAN, Pae GL-SNM, and Pae GP-SAN were 44.07%, 47.94% and 36.56%, respectively. The relatively low EE (less than 50%) observed for SGD-SAN, Pae GL-SNM, and Pae GP-SAN could be attributed to the high hydrophilicity of Pae.⁴¹ The DL of SGD-SAN, Pae GL-SNM and Pae GP-SAN were 5.54%, 10.70%, and 21.52%, respectively. The discrepancies in DL among SGD-SAN, Pae GL-SNM, and Pae GP-SAN are likely due to differences in the structural composition and size of the nanoparticles. SGD-SAN exhibited the lowest DL of Pae, because it encapsulated not only Pae but also other active ingredients, such as albiflorin, benzoylpaeoniflorin, and liquiritin.

In Vitro Drug Release

The in vitro release profiles of SGD-SAN, Pae GL-SNM, and Pae GP-SAN are shown in Figure 3. Similar drug release profiles were observed for the same Pae formulations in both the SGF and SIF. Compared to the Pae solution, Pae release from the three nano-assemblies (SGD-SAN, Pae GL-SNM, and Pae GP-SAN) exhibited significantly slower kinetic profiles. About 100% of Pae was rapidly released from the Pae solution within 2 h, whereas only 50%-75% of Pae was released from SGD-SAN, Pae GL-SNM, and Pae GP-SAN in SGF and SIF, which is likely due to the sustained diffusion barrier imposed by the nano-assemblies matrix and drug-carrier interactions.⁴² In addition, the Pae release from three nano-assemblies within 6 h in SGF followed the order: Pae GP-SAN > SGD-SAN > Pae GL-SNM, with Pae GP-SAN exhibiting the fastest release kinetics (74.61% at 2 h vs 61.54% for SGD-SAN and 53.20% for Pae GP-SAN), likely due to protein hydrolysis of GP in an acidic environment, which compromises the structural integrity of nano-assemblies and promotes payload liberation. Pae GL-SNM exhibited a relatively lower cumulative Pae release (53.20%) in SGF than in SIF (64.11%), which may be due to the suppression of Pae GL-SNM disassembly under low pH conditions, thereby maintaining the structural integrity of micelles and delaying drug release. The sustained release of Pae from the nano-assemblies may be beneficial for the oral absorption of Pae by facilitating prolonged mucosal interactions.⁴³

Figure 3 In vitro release profiles of Pae from Pae solution, SGD-SAN, Pae GL-SNM and Pae GP-SAN in SGF (A) and SIF (B) (mean ± SD, n=3).

Intestinal Absorption Characteristics of Nano-Assemblies

The intestinal perfusion studies in rats are the most reliable and cost-effective option among all methods for assessing intestinal drug absorption due to the high correlation between human and rat small intestinal permeability (r² = 0.8–0.95).^29,44 The in situ intestinal perfusion has been widely used to study the absorption of drugs in the intestine.^{18,28,29,39,45} Therefore, the absorptive behaviors of SGD-SAN, Pae GL-SNM, Pae GP-SAN, and Pae solution were compared by the in situ intestinal perfusion method, and gravimetry was employed instead of the classic phenol red method to determine perfusate volume changes in our study, owing to the partial intestinal absorption of phenol red.²⁹

 The K_a and P_eff obtained from the single-pass intestinal perfusion models are shown in Figure 4. Pae solution showed similar K_a and P_eff values in the jejunum and ileum. The K_a and P_eff values of SGD-SAN, Pae GL-SNM and Pae GP-SAN were higher than those of the Pae solution, indicating increased intestinal absorption of Pae by nano-assemblies. This may be explained as follows: firstly, GL in SGD-SAN and Pae GL-SNM may reduce drug efflux through the inhibition of P-gp and increase drug penetration through the intestinal epithelium by increasing permeability and decreasing the elasticity modulus of the cell membranes.^46,47 Secondly, the excellent mucoadhesion of nanoparticles to GI prolongs the drug residence time, possibly leading to increased drug absorption.⁴⁸ Finally, internalization of nanoparticles by epithelial cells may also contribute to intestinal absorption of Pae.⁴⁹ Among the three nano-assemblies, Pae GP-SAN showed the highest K_a and P_eff values in the ileum, suggesting the best intestinal absorption. The possible explanation is as follows: a key distinction between the jejunum and the ileum lies in the presence of specialized structures such as Peyer’s patches (PPs) in the latter.⁵⁰ The follicular-associated epithelial cells (FAE) are enriched in the PPs, where many membranous/microfold cells (M cells) reside in the FAE region.⁵¹ M cells are specialized antigen-transporting cells characterized by abundant vesicles, short microvilli, and low enzymatic activity, with a primary function of transporting antigens from the intestinal lumen to the subepithelial lymphoid tissue.⁵² Involved in antigen sampling, M cells have been extensively reported to possess strong transcytosis capacities to transport a wide range of particulate substances, including antigens, bacteria, viruses, and therapeutic nanoparticles. As an exogenous protein nanoparticle, Pae GP-SAN, with protein-rich composition, is likely to be recognized as antigenic substance and preferentially internalized by M cells, which likely accounts for its greater absorption in the ileum.⁴⁹

Figure 4 The (A) and (B) of Pae from Pae solution, SGD-SAN, Pae GL-SNM and Pae GP-SAN in the jejunum and ileum (mean ± SD, n=3; *P < 0.05, **P < 0.01 vs Pae solution; #P < 0.05, ##P < 0.01 vs SGD-SAN; &P < 0.05 vs Pae GL-SNM).

Pharmacokinetics of Nano-Assemblies

The plasma concentration-time curves of Pae after oral administration of Pae solution, SGD-SAN, Pae GL-SNM, and Pae GP-SAN are shown in Figure 5, and the main pharmacokinetic parameters are presented in Table 4. In agreement with our previous findings,¹⁸ Pae solution demonstrated poor oral absorption, as evidenced by its low C_max (964.89 ± 128.81 ng/mL) and AUC_0-t (5676.14 ± 311.61 ng/mL·h), which could be attributed to its high hydrophilicity, P-gp mediated efflux, and hydrolysis in the intestine.⁵³ Compared with the Pae solution, SGD-SAN exhibited a relatively higher plasma concentration of Pae at all time points following oral administration. The C_max (1907.48 ± 245.22 ng/mL) and AUC_0-t (8241.69 ± 944.62 ng/mL·h) of Pae after oral administration of SGD-SAN were significantly higher than those of Pae solution, indicating significant enhancement in the oral bioavailability of Pae. Furthermore, the results also suggested that the bioavailability improvement of Pae by oral administration of SGD may be related to the formation of SGD-SAN during decoction. The bioavailability improvement of Pae by SGD-SAN can be attributed to the prolonged drug release and the increased intestinal absorption. In addition, both the bioadhesive properties and the overall transmembrane capacity of nanoparticles also probably contribute to the enhanced Pae absorption.^42,54

Table 4 The Pharmacokinetic Parameters of Pae Following Oral Administration of Pae Solution, SGD-SAN, Pae GL-SNM and Pae GP-SAN (Mean ± SD, n=6)

Figure 5 Plasma concentration-time curves of Pae following oral administration of Pae solution, SGD-SAN, Pae GL-SNM and Pae GP-SAN (mean ± SD, n=6).

Notably, the bioavailability of Pae was also significantly improved by the oral administration of Pae GL-SNM and Pae GP-SAN, as evidenced by the higher AUC_0-t (9362.52 ± 854.15 ng/mL·h for Pae GL-SNM and 11209.01 ± 2093.72 ng/mL·h for Pae GP-SAN). The same reason as proposed above with SGD-SAN can be employed to interpret the bioavailability improvement of Pae by the oral administration of Pae GL-SNM and Pae GP-SAN. Moreover, Pae GP-SAN showed significantly higher AUC_0-t as compared to SGD-SAN, which could be explained by the increased intestinal absorption in the ileum. Both intestinal perfusion and pharmacokinetic studies demonstrated that SGD-SAN, Pae GL-SNM, and Pae GP-SAN significantly enhanced the oral absorption of Pae, with Pae GP-SAN exhibiting the best oral absorption. These results suggested that nano-assemblies, particularly protein-based GP-SAN, overcome the absorption barriers of Pae through ileum-targeted uptake, sustained release, and nanoparticle bioadhesion, providing a structural foundation for SGD’s bioavailability-enhancing effect.

Potential Formation Mechanisms of Nano-Assemblies

As is well known, urea and Tween 20 are capable of disrupting hydrogen bonds and hydrophobic interactions, respectively, while NaCl can reduce electrostatic interactions;²⁰ these chemical interference agents have been widely used to study the mechanism of nanocomplex formation.^33,34 To gain more insights into which dominant driving forces are present, we investigated the particle size change of SGD-SAN, Pae GP-SAN, and Pae GL-SNM after adding urea, NaCl, and Tween 20. As shown in Figure 6A, increasing concentrations of urea significantly increased the particle size of Pae GL-SNM, while exerting no effect on that of SGD-SAN and Pae GP-SAN, indicating that hydrogen bonds may not play a dominant role in driving the assembly or maintaining the structural stability of SGD-SAN and Pae GP-SAN, but play a non-negligible role in either the formation or, more notably, the structural stability of Pae GL-SNM.

Figure 6 The particle size change of SGD-SAN, Pae GP-SAN, and Pae GL-SNM after adding urea (A), NaCl (B), and Tween 20 (C).

Figure 6B showed that increasing concentrations of NaCl exerted no effect on the particle size of SGD-SAN, led to a slight increase in the particle size of Pae GP-SAN, and caused a significant increase in the particle size of Pae GL-SNM. Notably, when the NaCl concentration was equal to or higher than 0.05 M, Pae GL-SNM underwent obvious gelation. The absence of particle size change of SGD-SAN with increasing NaCl concentrations indicates that its stable dispersion is completely independent of electrostatic interactions. The slight particle size enlargement of Pae GP-SAN implies a weak reliance on electrostatic interactions. The significant particle size increase and subsequent gelation (at NaCl ≥ 0.05 M) of Pae GL-SNM demonstrate its strong dependence on electrostatic interactions.

Increasing concentrations of Tween 20 exerted a significant impact on the particle sizes of SGD-SAN, Pae GP-SAN, and Pae GL-SNM, but with distinct concentration-dependent trends (Figure 6C). The particle size increased significantly with rising Tween 20 concentration and tended to plateau (~800 nm) after the concentration reached 5 mM, while the particle size of Pae GP-SAN remained unchanged initially and then increased significantly (~1600 nm) when the Tween 20 concentration reached 10 mM. The results indicated that hydrophobic interactions play a critical role in maintaining the stability of SGD-SAN and Pae GP-SAN. For Pae GL-SNM, the particle size increased slightly to approximately 200 nm at Tween 20 concentrations below 2 mM, followed by a significant decrease to approximately 20 nm at and above 5 mM. This is because at low concentrations, Tween 20 only partially inserts into the Pae GL-SNM, causing a slight size increase due to structural loosening or shell expansion. However, once a critical concentration is exceeded, it sufficiently disrupts the hydrophobic interactions to disassemble the original micelles and form smaller mixed or Tween 20-dominated micelles, resulting in a significant size reduction.

Based on the above results, we can draw the following conclusions: hydrophobic interactions serve as the common core force across all nano-assemblies, while their dependencies on other intermolecular forces differ significantly. Specifically, hydrophobic interactions act as the core driving force for the formation of Pae GL-SNM, with hydrogen bonds and electrostatic interactions jointly assisting in maintaining the structural stability of the formed micelles; for Pae GP-SAN, its formation is driven by a synergistic effect dominated by hydrophobic interactions and supplemented by electrostatic interactions; while for SGD-SAN, hydrophobic interactions may alone constitute the core force supporting its structural stability.

Conclusions

In this study, three nano-assemblies of different components from SGD (SGD-SAN, Pae GL-SNM, and Pae GP-SAN) were compared in vitro and in vivo, with particular emphasis on their differential effects on Pae bioavailability. Three nano-assemblies showed similar spherical shape, but exhibited significant differences in particle size (133 nm for SGD-SAN, 154 nm for Pae GL-SNM, and 184 nm for Pae GP-SAN), PDI, zeta potential, EE, and DL (5.54% for SGD-SAN, 10.70% for Pae GL-SNM, and 21.52% for Pae GP-SAN). While hydrophobic interactions act as the common core force driving the formation of all nano-assemblies, their dependencies on other intermolecular forces vary remarkably. SGD-SAN, Pae GL-SNM, and Pae GP-SAN exhibited sustained Pae release (50–75% over 12 h vs 100% for the Pae solution in 2 h). In situ intestinal perfusion experiments in rats showed significantly higher effective permeability coefficients (P_eff) for all nano-assemblies than the Pae solution, with Pae GP-SAN exhibiting the highest ileal absorption, which may be attributed to preferential M-cell uptake facilitated by its larger size and protein-rich composition. Pharmacokinetic studies further confirmed superior performance of Pae GP-SAN with the highest AUC_0-t (11209.01 ± 2093.72 ng/mL·h) and C_max (2896.04 ± 255.01 ng/mL), representing 2.0-fold and 3.0-fold increases over Pae solution (5676.14 ± 311.61 ng/mL·h and 964.89 ± 128.81 ng/mL), respectively. Additionally, Pae GL-SNM and SGD-SAN significantly enhanced Pae bioavailability, with AUC_0-t increased by 65% and 45%, respectively. These results suggested that nano-assemblies, particularly protein-based Pae GP-SAN, overcome the absorption barriers of Pae through ileum-targeted uptake, sustained release, and nanoparticle bioadhesion, thereby providing a structural basis for SGD’s ability to enhance Pae bioavailability. The comprehensive comparisons elucidated the structure-function relationships underlying SGD-mediated improvements in Pae absorption. However, the reasons for the difference in Pae absorption among the three nano-assemblies remain unclear. In particular, no direct experimental evidence has been provided to support ileal M-cell uptake proposed as a key mechanism for Pae GP-SAN’s superior absorption. Further studies should be performed to clarify their mechanisms for enhancing the oral absorption of Pae.

Acknowledgments

This work was partially supported by the National Natural Science Foundation of China (82260848), the Natural Science Foundation of Jiangxi Province (20232BAB216139), – the Technology Innovation Team of Jiangxi University of Traditional Chinese Medicine (CXTD22006)-, and the Innovation Special Fund for graduate students of Jiangxi University of Chinese Medicine (202510412257).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors have no relevant financial or non-financial interests to disclose.

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