Development and Bioequivalence of 3D-Printed Medication at the Point-of-Care: Bridging the Gap Toward Personalized Medicine.

Personalized medicine is currently hampered by the lack of flexible drug formulations. Especially for pediatric patients, manual compounding of personalized drug formulations by pharmacists is required. Three-Dimensional (3D) printing of medicines, which enables small-scale manufacturing at the point-of-care, can fulfill this unmet clinical need. This study investigates the feasibility of developing a 3D-printed tablet formulation at the point-of-care which complies to quality requirements for clinical practice, including bioequivalence. Development, manufacturing, and quality control of the 3D-printed tablets was performed at the manufacturing facility and laboratory of the department of Clinical Pharmacy and Toxicology at Leiden University Medical Center. Sildenafil was used as a model drug for the tablet formulation. Along with the 3D-printed tablets a randomized, an open-label, 2-period, crossover, single-dose clinical trial to assess bioequivalence was performed in healthy adults. Bioequivalence was established if areas under the plasma concentration curve from administration to the time of the last quantifiable concentration (AUC0-t ) and maximum plasma concentration (Cmax ) ratios were within the limits of 80.00-125.00%. The manufacturing process provided reproducible 3D-printed tablets that adhered to quality control requirements and were consequently used in the clinical trial. The clinical trial was conducted in 12 healthy volunteers. The 90% confidence intervals (CIs) of both AUC0-t and Cmax ratios were within bioequivalence limits (AUC0-t 90% CI: 87.28-104.14; Cmax 90% CI: 80.23-109.58). For the first time, we demonstrate the development of a 3D-printed tablet formulation at the point-of-care that is bioequivalent to its marketed originator. The 3D printing of personalized formulations is a disruptive technology for compounding, bridging the gap toward personalized medicine.

3D Printing of Pediatric Medication: The End of Bad Tasting Oral Liquids?-A Scoping Review.

3D printing of pediatric-centered drug formulations can provide suitable alternatives to current treatment options, though further research is still warranted for successful clinical implementation of these innovative drug products. Extensive research has been conducted on the compliance of 3D-printed drug products to a pediatric quality target product profile. The 3D-printed tablets were of particular interest in providing superior dosing and release profile similarity compared to conventional drug manipulation and compounding methods, such as oral liquids. In the future, acceptance of 3D-printed tablets in the pediatric patient population might be better than current treatments due to improved palatability. Further research should focus on expanding clinical knowledge, providing regulatory guidance and expansion of the product range, including dosage form possibilities. Moreover, it should enable the use of diverse good manufacturing practice (GMP)-ready 3D printing techniques for the production of various drug products for the pediatric patient population.

Eradication of meticillin-resistant Staphylococcus aureus from human skin by the novel LL-37-derived peptide P10 in four pharmaceutical ointments.

Skin bacterial colonization/infection is a frequent cause of morbidity in patients with chronic wounds and allergic/inflammatory skin diseases. This study aimed to develop a novel approach to eradicate meticillin-resistant Staphylococcus aureus (MRSA) from human skin. To achieve this, the stability and antibacterial activity of the novel LL-37-derived peptide P10 in four ointments was compared. Results indicate that P10 is chemically stable and antibacterial in hypromellose gel and Softisan-containing cream, but not in Cetomacrogol cream (with or without Vaseline), at 4 °C for 16 months. Reduction in MRSA counts on Leiden human epidermal models (LEMs) by P10 in hypromellose gel was greater than that of the peptide in Cetomacrogol cream or phosphate buffered saline. P10 did not show adverse effects on LEMs irrespective of the ointment used, while Cetomacrogol with Vaseline and Softisan cream, but not hypromellose gel or Cetomacrogol cream, destroyed MRSA-colonized LEMs. Taking all this into account, P10 in hypromellose gel dose-dependently reduced MRSA colonizing the stratum corneum of the epidermis as well as biofilms of this bacterial strain on LEMs. Moreover, P10 dose-dependently reduced MRSA counts on ex-vivo human skin, with P10 in hypromellose gel being more effective than P10 in Cetomacrogol and Softisan creams. P10 in hypromellose gel is a strong candidate for eradication of MRSA from human skin.

Transcriptome Signature Reversion as a Method to Reposition Drugs Against Cancer for Precision Oncology.

Transcriptome signature reversion (TSR) has been hypothesized as a promising method for discovery and use of existing noncancer drugs as potential drugs in the treatment of cancer (i.e., drug repositioning, drug repurposing). The TSR assumes that drugs with the ability to revert the gene expression associated with a diseased state back to its healthy state are potentially therapeutic candidates for that disease. This article reviews methodology of TSR and critically discusses key TSR studies. In addition, potential conceptual and computational improvements of this novel methodology are discussed as well as its current and possible future application in precision oncology trials.

The impact of estimated tumour purity on gene expression-based drug repositioning of Clear Cell Renal Cell Carcinoma samples.

To find new potentially therapeutic drugs against clear cell Renal Cell Carcinoma (ccRCC), within drugs currently prescribed for other diseases (drug repositioning), we previously searched for drugs which are expected to bring the gene expression of 500 + ccRCC samples from The Cancer Genome Atlas closer to that of healthy kidney tissue samples. An inherent limitation of this bulk RNA-seq data is that tumour samples consist of a varying mixture of cancerous and non-cancerous cells, which influences differential gene expression analyses. Here, we investigate whether the drug repositioning candidates are expected to target the genes dysregulated in ccRCC cells by studying the association with tumour purity. When all ccRCC samples are analysed together, the drug repositioning potential of identified drugs start decreasing above 80% estimated tumour purity. Because ccRCC is a highly vascular tumour, attributed to frequent loss of VHL function and subsequent activation of Hypoxia-Inducible Factor (HIF), we stratified the samples by observed activation of the HIF-pathway. After stratification, the association between estimated tumour purity and drug repositioning potential disappears for HIF-activated samples. This result suggests that the identified drug repositioning candidates specifically target the genes expressed by HIF-activated ccRCC tumour cells, instead of genes expressed by other cell types part of the tumour micro-environment.

Personalised drug repositioning for Clear Cell Renal Cell Carcinoma using gene expression.

Reversal of cancer gene expression is predictive of therapeutic potential and can be used to find new indications for existing drugs (drug repositioning). Gene expression reversal potential is currently calculated, in almost all studies, by pre-aggregating all tumour samples into a single group signature or a limited number of molecular subtype signatures. Here, we investigate whether drug repositioning based on individual tumour sample gene expression signatures outperforms the use of tumour group and subtype signatures. The tumour signatures were created using 534 tumour samples and 72 matched normal samples from 530 clear cell renal cell carcinoma (ccRCC) patients. More than 20,000 drug signatures were extracted from the CMAP and LINCS databases. We show that negative enrichment of individual tumour samples correlated (Spearman's rho = 0.15) much better with the amount of differentially expressed genes in drug signatures than with the tumour group signature (Rho = 0.08) and the 4 tumour subtype signatures (Rho 0.036-0.11). Targeted drugs used against ccRCC, such as sirolimus and temsirolimus, which could not be identified with the pre-aggregated tumour signatures could be recovered using individual sample analysis. Thus, drug repositioning can be personalized by taking into account the gene expression profile of the individual's tumour sample.

The possibility of obtaining marketing authorization of orphan pharmaceutical compounding preparations: 3,4-DAP for Lambert-Eaton Myasthenic Syndrome.

BACKGROUND: Pharmaceutical compounding preparations, produced by (hospital) pharmacies, usually do not have marketing authorization. As a consequence, some of these pharmaceutical compounding preparations can be picked-up by a pharmaceutical company to obtain marketing authorization, often leading to price increases. An example is the 3,4-diaminopyridine slow release (3,4-DAP SR) tablets for Lambert-Eaton Myasthenic Syndrome (LEMS). In 2009 marketing authorization was given for the commercial immediate release phosphate salt of the drug, including a fifty-fold price increase compared to the pharmaceutical compounding preparation. Obtaining marketing authorization for 3,4-DAP SR by academia might have been a solution to prevent this price increase. To determine whether the available data of a pharmaceutical compounding preparation with long-term experience in regular care are adequate to obtain marketing authorization, 3,4-DAP SR is used as a case study. METHODS: A retrospective qualitative case-study was performed. Initially, document analysis was executed by collecting the required data for marketing authorization in general and whether data of Firdapse® and 3,4-DAP SR met these requirements. Secondly, the (non-) available data of the two formulations were compared with each other to determine the differences in availability. RESULTS: At the time of approval, almost all data were available for both Firdapse® and 3,4-DAP SR. Conversely, much of the data used for the approval of Firdapse® originated from the 3,4-DAP immediate release (3,4-DAP IR) formulation. Only two bioequivalence studies and one pharmacology safety study was performed with Firdapse® before marketing authorization application. CONCLUSIONS: In conclusion, at time Firdapse® obtained approval, the data available did not differ substantially from 3,4-DAP SR, indicating that approval with 3,4-DAP SR would have been possible. We make a plea for approval of orphan medicinal products developed and manufactured by academic institutions as to keep utilization of these products affordable.

Unlicensed pharmaceutical preparations for clinical patient care: Ensuring safety.

Most medicinal products dispensed to patients have marketing authorization (MA) to ensure high quality of the product, safety, and efficacy. However, in daily practice, to treat patients adequately, there is a medical need for drugs that do not hold MA. To meet this medical need, medicinal products are used in clinical care without MA (unlicensed), such as products prepared by (local) pharmacies: the pharmaceutical preparations. Three types of pharmaceutical preparations are distinguished: (i) reconstitution in excess of summary of product characteristics; (ii) adaptation of a licensed medicinal product (outside its official labeling); (iii) medicinal products from an active pharmaceutical ingredient. Although unlicensed, patients may expect the same quality for these unlicensed pharmaceutical preparations as for the licensed medicinal products. To assure this quality, a proper risk-benefit assessment and proper documentation in (centralized) patient registries and linking to a national pharmacovigilance database should be in place. Based on a risk assessment matrix, requirements for quality assurance can be determined, which has impact on the level of documentation of a pharmaceutical preparation. In this paper, the approach for good documentation including quality assurance and benefit-risk assessment will be discussed and possibilities for patient registries are described to make these crucial preparations available for regular patient care. KEY POINTS Ensuring pharmaceutical quality and performing a proper benefit-risk assessment will guarantee safe use of pharmaceutical preparations. Good documentation of (ultra-)orphan treatments can be collected in centralized patient registries and should be combined with existing information in (inter)national databases and self-reflection of patients. Linking patient registries to a centralized database for adverse drug events is highly recommended as it increases safety control of the (ultra) orphan pharmaceutical preparations.

Aggregated N-of-1 trials for unlicensed medicines for small populations: an assessment of a trial with ephedrine for myasthenia gravis.

BACKGROUND: Inexpensive medicines with a long history of use may currently be prescribed off-label for rare indications. Reimbursement is at the discretion of health insurance companies, and may be unpredictable. The example addressed was ephedrine as add-on treatment for myasthenia gravis. Stakeholders from academia, a patient organization, the Dutch National Health Care Institute (NHCI) and Dutch Medicines Evaluation Board (MEB) advised on the trial design. The NHCI and MEB agreed to provide scientific advice on the suitability of the evidence generated by the trial, for regulatory decisions. This paper describes the feasibility of the trial and the utility of its aggregated results. RESULTS: The trialists experienced the trial as feasible. Retrospective interviews showed that the trial as performed was acceptable to patients. The treatment effect in the primary outcome measure, muscle strength, was statistically significant when inferred to the population level, though the effect size was modest. Secondary outcomes were statistically significant in a preplanned, fixed effects analysis within the four patients. The NHCI advised that it could potentially make reimbursement decisions based on the Fitting Evidence framework, should the trialists decide to apply for reimbursement. The MEB advised that for a licensing decision, the N-of-1 design is a last-resort option for demonstrating treatment benefit in a rare disease. N-of-1 trials alone do not provide enough evidence on potential risk. The MEB found the current trial inconclusive. It suggested doing a 2-armed trial of longer duration, possibly with a different outcome measure (postponement of corticosteroid use). It suggested engaging a consultancy or commercial sponsor, should the trialists decide to seek market authorization of the drug. CONCLUSIONS: In theory, evidence from aggregated N-of-1 trials is suitable for use in licensing and reimbursement decisions. The current example illustrates differences in interpretation of N-of-1 results by health authorities. In the era of personalized medicine, consensus is required on the interpretation of data from study designs geared to small groups. Demonstrating effectiveness of inexpensive medicines in small populations may require involvement of non-commercial parties, to preserve affordability.

Ephedrine treatment for autoimmune myasthenia gravis.

We studied the effect and safety of ephedrine as add-on treatment for patients with myasthenia gravis with acetylcholine receptor antibodies (AChR MG), who do not sufficiently respond to standard treatment. Four patients with AChR MG were included in a placebo-controlled, double-blind, and randomised, multiple crossover series of n-of-1 trials. Each n-of-1 trial consisted of 3 cycles, in which two 5-day intervention periods were followed by 2 days washout. In each cycle, ephedrine 50 mg daily in 2 doses was compared with placebo in the alternate treatment period. Primary outcome was a change in QMG score. Add-on treatment with ephedrine compared with placebo improved QMG score by 1.0 point (95% confidence interval 0.21-1.79), which was significant for the group of trial patients as well as for the population treatment effect. Ephedrine also showed a significant trial average treatment effect for all secondary outcomes, improving MG Composite by 2.7, MG-ADL by 1.0 and VAS score for muscle strength by 1.1. Adverse events were mild and included palpitations, tremor and restlessness. Although all ECGs were normal, ephedrine prolonged the corrected QT interval. Ephedrine as add-on treatment for myasthenia gravis resulted in a small but consistent reduction of symptoms and weakness in patients with moderate disease severity.

Antimicrobial Peptide P60.4Ac-Containing Creams and Gel for Eradication of Methicillin-Resistant Staphylococcus aureus from Cultured Skin and Airway Epithelial Surfaces.

We previously found the LL-37-derived peptide P60.4Ac to be effective against methicillin-resistant Staphylococcus aureus (MRSA) on human epidermal models (EMs). The goal of this study was to identify the preferred carrier for this peptide for topical application on skin and mucosal surfaces. We prepared P60.4Ac in three formulations, i.e., a water-in-oil cream with lanolin (Softisan 649), an oil-in-water cream with polyethylene glycol hexadecyl ether (Cetomacrogol), and a hydroxypropyl methylcellulose (hypromellose) 4000 gel. We tested the antimicrobial efficacy of the peptide in these formulations against mupirocin-resistant and -sensitive MRSA strains on EMs and bronchial epithelial models (BEMs). The cytotoxic effects of formulated P60.4Ac on these models were determined using histology and WST-1 and lactate dehydrogenase assays. Moreover, we assessed the stability of the peptide in these formulations with storage for up to 3 months. Killing of MRSA by P60.4Ac in the two creams was less effective than that by P60.4Ac in the hypromellose gel. In agreement with those findings, P60.4Ac in the hypromellose gel was highly effective in eradicating the two MRSA strains from EMs. We found that even 0.1% (wt/wt) P60.4Ac in the hypromellose gel killed >99% of the viable planktonic bacteria and >85% of the biofilm-associated bacteria on EMs. Hypromellose gels containing 0.1% and 0.5% (wt/wt) P60.4Ac effectively reduced the numbers of viable MRSA cells from BEMs by >90%. No cytotoxic effects of P60.4Ac in the hypromellose gel with up to 2% (wt/wt) P60.4Ac on keratinocytes in EMs and in the hypromellose gel with up to 0.5% (wt/wt) P60.4Ac on epithelial cells in BEMs were observed. High-performance liquid chromatography analysis showed that P60.4Ac was stable in the Softisan cream and the hypromellose gel but not in the Cetomacrogol cream. We conclude that P60.4Ac formulated in hypromellose gel is both stable and highly effective in eradicating MRSA from colonized EMs and BEMs.

Ephedrine as add-on therapy for patients with myasthenia gravis: protocol for a series of randomised, placebo-controlled n-of-1 trials.

INTRODUCTION: Myasthenia gravis (MG), a rare neuromuscular disease, is often initially treated using acetylcholinesterase inhibitors. Patients who do not respond adequately depend on the use of corticosteroids or other immunosuppressive medication, but these may have serious side effects. Clinical observations suggest that ephedrine can diminish, postpone or even prevent the need for immunosuppressive therapy when added to acetylcholinesterase inhibitors or low-dose prednisone. In the Netherlands, ephedrine is not licensed for MG nor is reimbursement guaranteed. MG is a rare condition, and ephedrine might be indicated only in a subset of patients. Thus, randomised controlled trials comparing large groups are difficult to conduct. We, therefore, aim to aggregate data from a small series of n-of-1 trials (also known as single patient trials) to assess the effect of ephedrine as add-on treatment for MG. METHODS AND ANALYSIS: Single-centre, placebo-controlled, double-blind, randomised, multiple crossover n-of-1 studies in 4 adult patients with generalised MG who show inadequate improvement on pyridostigmine and/or immunosuppressive drugs. Each n-of-1 trial has 3 cycles of two 5-day intervention periods. TREATMENT: 25 mg ephedrine or placebo, twice daily. MAIN OUTCOME MEASURE: Quantitative Myasthenia Gravis (QMG) test. STATISTICAL ANALYSIS: fixed effects linear model for QMG for all patients combined. SECONDARY OUTCOME MEASURES: Clinical: effects on MG-Composite and MG-Activities of Daily Living (MG-ADL) scales; QMG at individual level; adverse events. Acceptability of trial design: number of patients eligible and enrolled; number of treatment cycles completed; patients' and caregivers' experiences. ETHICS AND DISSEMINATION: This study was approved by the Medical Ethics Committee of Leiden University Medical Center, No. P14.108. Results of the trial will be reported in a peer-reviewed publication. Regulatory stakeholders will comment on the suitability of the trial for market authorisation and reimbursement purposes. TRIAL REGISTRATION NUMBER: This study is registered under EudraCT number 2014-001355-23, protocol no. 40960, V.1.0, registration date 27 March 2014.

Development of a nose cream containing the synthetic antimicrobial peptide P60.4Ac for eradication of methicillin-resistant Staphylococcus aureus carriage.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing problem, and current treatment options are suboptimal. Nasal carriage of MRSA is a major risk factor for infection, but nasal eradication strategies are increasingly considered to be insufficiently effective. In this study, a water-in-oil cream formulation was developed for nasal application with an antimicrobial peptide, P60.4Ac, aimed at the eradication of MRSA carriage. Quality control of the cream included the measurement of the content and release of the peptide by a validated high-performance liquid chromatography method. Stability of the peptide in the formulation was investigated including the evaluation of the effect of stress conditions. Preliminary shelf-life study of the drug formulation demonstrated that the peptide is stable in the formulation at least for 5 months. Microbial-killing assays with MRSA LUH14616 as a target demonstrated the dose-dependent antimicrobial activity of the peptide formulation.

Limited antitumor-effect associated with toxicity of the experimental cytotoxic drug cyclopentenyl cytosine in NOD/scid mice with acute lymphoblastic leukemia.

The experimental cytotoxic drug cyclopentenyl cytosine (CPEC) is a non-competitive inhibitor of the enzyme cytidine triphosphate (CTP) synthethase. We evaluated the in vitro and in vivo antitumor activity of CPEC on human acute lymphoblastic leukemia (ALL) cell lines. CPEC displayed anti-leukemic activity with IC50 (after 3 days of incubation) ranging from 6 to 15 nM. Subsequently the in vivo activity of CPEC against primary human ALL was evaluated in a xenogeneic model of human ALL using NOD/scid mice inoculated with primary human ALL cells. In the model, only a marginal anti-leukemic activity was observed at 1.5 mg kg(-1) (5 days per week) and 5 mg kg(-1) (2 days per week), however, this activity was associated with severe systemic toxicity. The observed toxicity was not specific for the NOD/scid model, as toxicity at comparable treatment intensity was also observed in Balb/c mice. In conclusion, although CPEC showed antitumor activity against human ALL cells in vitro, its activity in the in vivo human leukemia model was only marginal and accompanied by severe toxicity.

Radiopharmaceutical management of 90Y/111In labeled antibodies: shielding and quantification during preparation and administration.

BACKGROUND: The combined application of potent beta-emitting isotopes for therapy with remitting isotopes for scintigraphy requires a profound regimen concerning team member safety and radionuclide quantification. METHODS: We have developed materials and methods for a proper and easy manipulation of 90Y during preparation and administration of 90Y/111In pharmaceuticals used for radioimmunotherapy. RESULTS: The efficacy of the shielding measures is documented. Protocols for the calibration of gamma-dose calibrators with respect to 90Y are extended to the assessment of quench-corrected liquid scintillation counting of 90Y. The contribution of 90Y backscatter to 111 In counting is quantified. Newly developed shielding equipment allows an adequate administration of relatively large volumes (100 ml) of 90Y/111In labeled pharmaceuticals to patients. CONCLUSIONS: The procedures described combine pharmaceutical (Good Manufacturing Practice) and radiation safety requirements with an accurate logging of relevant data.

Preparation, radiochemical purity control and stability of 99mTc-mertiatide (Mag-3).

BACKGROUND: Scintigraphic image analysis of 99mTc-mertiatide (Mag-3, mercaptoacetyltriglycine) clearance provides the determination of the blood flow, the tubular transit time and the excretion as well from both kidneys. Radiopharmaceutical routine recommends a radiochemical purity control before administration of the product to a patient. The main objective of this study is to develop a Mag-3 labeling procedure that fits better than the previous one in our daily routine production of radiopharmaceuticals. METHODS: Increasing proportions of 99mTc-Mag-3 were measured during the heating and cooling steps of the Mag-3 labeling procedure. HPLC analysis was used to confirm the results of a rapid radiochemical quality control assay on standard ITLC-SG paper. RESULTS: The reconstitution time takes 20-25 minutes from the harvest of pertechnetate to a ready-for-use calibrated patient syringe. The HPLC profile of 99mTc-Mag-3 including its minor impurities remains unchanged for 24-48 hours after reconstitution. CONCLUSIONS: The application of a programmable Peltier-directed device for heating/cooling provides a better control of the temperature course. The procedure proposed fully meets the labeling criteria recommended by the supplier and can be performed with a minimum of attention within a time-span that we formerly needed for solely the radiochemical purity control assay. Moreover, 99mTc-Mag-3 prepared in this way seems to be considerably more stable than mentioned in the manufacturer's instructions.

Cardiotoxicity of cytotoxic drugs.

Cardiotoxicity is a well-known side effect of several cytotoxic drugs, especially of the anthracyclines and can lead to long term morbidity. The mechanism of anthracycline induced cardiotoxicity seems to involve the formation of free radicals leading to oxidative stress. This may cause apoptosis of cardiac cells or immunologic reactions. However, alternative mechanisms may play a role in anthracycline induced cardiotoxicity. Cardiac protection can be achieved by limitation of the cumulative dose. Furthermore, addition of the antioxidant and iron chelator dexrazoxane to anthracycline therapy has shown to be effective in lowering the incidence of anthracycline induced cardiotoxicity. Other cytotoxic drugs such as 5-fluorouracil, cyclophosphamide and the taxoids are associated with cardiotoxicity as well, although little is known about the possible mechanisms. Recently, it appeared that some novel cytotoxic drugs such as trastuzumab and cyclopentenyl cytosine also show cardiotoxic side effects.