Cristiana Bernini, Autore a NCF International

Oncology and antibiotics for Merck

Business

Cristiana Bernini

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7 April 2015

Merck (MSD) has recently closed two different deals to strengthen its position in the oncology and antibiotics segments, respectively. The US-based big pharma has acquired the Swiss privately held biotechnology company OncoEthix, focused in oncology drug development. Its main investigational product is OTX015, a novel oral BET (bromodomain) inhibitor currently in Phase Ib studies for the treatment of hematological malignancies and advanced solid tumors.

BET proteins play a pivotal role in regulating the transcription of key regulators of cancer cell growth and survival. An international, open-label phase I study evaluating OTX015 in five different solid tumors was initiated in November 2014.

Upfront payment has been established up to $110 million, according to Merck. Additional milestone payments of up to $265 million are contingent the achievement of clinical and regulatory endpoints.

The second acquisition involves antibiotic specialist Cubist Pharmaceuticals, for a total sum of $8.4 billions. Cubist, founded in 1992, is one of the few companies active in the research and development of new antibiotic treatments. Its more advanced product is daptomycin (for injection), already approved for complicated skin and skin structure infections caused by certain bacteria and in Staphylococcus aureus infections of the bloodstream. The company plans to launch several new antibiotic drugs by 2020.

Acquisition to strengthen communication services

Business

Cristiana Bernini

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7 April 2015

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Icon, the global provider of outsourced development services, has announced the intention to acquire MediMedia Pharma Solutions. The deal shall count for $120 million.

MediMedia Pharma Solutions, a division of MediMedia USA, and is owned by Vestar Capital Partners.

The acquired company includes MediMedia Managed Markets, leading provider of strategic payer-validated market access solutions, and Complete Healthcare Communications, one of the leading medical and scientific communication agencies.

According to Icon, the acquisition strengthens its expertise in scientific communications and market access in order to create an integrated scientific communications and market access solution.

Acquisition in rare disease

Business

Cristiana Bernini

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7 April 2015

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Shire and NPS Pharmaceuticals, a rare disease-focused biopharmaceutical company, closed a merger agreement for a total of approximately $5.2 billion. Teduglutide for injection is NPS’s first product approved in the US and Europe to treat adults with short bowel syndrome (SBS) who are dependent on parenteral support. The second product, rhPTH-83, is under registration phase for the treatment of hypoparathyroidism (HPT). An ongoing phase IIa study is evaluating NPS’s lead pipeline candidate NPSP795 for the treatment of adults with autosomal dominant hypocalcemia.

The deal strengthens Shire’s focus on rare diseases.

Pfizer to enter biosimilars’ market

Business

Cristiana Bernini

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7 April 2015

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Pfizer has announced the intention to acquire Hospira, a leading company in the production of biosimilar drugs, which are the generic copies of biotech drugs. The total value of the deal is about $17 billion, the companies said.

The operation may open new strategic frontiers for Pfizer business, as biosimilars will expand their market positions upon the expiring of original patents. A biosimilar drug typically costs 20-30 percent less than the original.

Hospira is waiting for the approval from the FDA for its copy of Johnson & Johnson’s blockbuster arthritis treatment infliximab.

Oncology pipeline to extend to second clinical candidate

Business

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16 December 2014

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The strategic oncology partnership between Oxford BioTherapeutics and Berlin Chemie/Menarini Group has designated its second clinical development candidate: a novel antibody-drug conjugate (ADC) targeting Non-Hodgkin’s B-Cell Lymphoma and solid tumours, including some subsets of breast cancers. The ADC is currently undergoing formal regulatory enabling studies to support an application for the future clinical phase. The ADC candidate has completed in vivo proof-of-concept in several solid and liquid tumour models and exploratory toxicology testing.

Digiline Booklet now available from Atlantic Zeiser

Packaging

Cristiana Bernini

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9 December 2014

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Digiline Booklet now available from Atlantic Zeiser

Headquartered in Emmingen, in the Southern German region of Baden Württemberg, Atlantic Zeiser manages eleven facilities overseas and its products are distributed in 50 different countries thanks to a network of sales agents and service partners.

The company operates worldwide with the Card Systems and Banknotes divisions, set to address the banking and finance sector, and with the Packaging division that develops specific solutions for the pharmaceutical and cosmetics industries. Focused on the track and trace and digital printing most innovative systems, the latter of these has recently introduced a new entry in its world-renown Digiline product portfolio. Designed in order to meet the pharma segment’s most rigorous requirements, Digiline Booklet is an integrated system aimed at coding up to 4 millimeters thick booklet labels in an audit-proof process that can constantly be verified by an inspection camera. Able to ensure economic efficiency even with the shortest print runs and with completely different booklet formats, Digiline Booklet was developed for coding all those labels that are to be used in clinical drug trials.

Atlantic Zeiser believes the request for such technologies is about to rocket, together with international regulations and safety requirements that, increasing with time, will force the industry and laboratories to adopt thicker booklet labels to include a constantly growing number of mandatory information and contents. According to the German producer, «thermal transfer printers are no longer able to process the peaks and troughs associated with booklet labels» and this could cause serious problems since «printing defects can jeopardize not only market authorization and substantial investments, but also human lives». This is also one of the reasons why Atlantic Zeiser chose to leverage the contactless drop-on-demand inkjet technology, that can allow the Digiline Booklet to achieve an enhanced print quality and consistency, regardless of such issues as the font size, whilst on the other hand reducing operating cost by eliminating ink ribbons and print bars. A patented sensor-controlled transport system ensures that «the first incoming label of the roll is printed without any additional splicing of blank ribbon», as the Emmingen-based firm stated, and this way «no blank labels can enter circulation, auxiliary tapes and working time are avoided, and expensive label waste is ruled out». An excellent print quality is also guaranteed in case of machine stops, while an integrated camera was specifically designed for an in-depth inspection of all the processed labels. «Furthermore», Atlantic Zeiser added, «a side register camera compensates the sidewise print position of each individual label and thus contributes to precise sidewise pint accuracy», despite any possible inaccurate label positioning on the ribbon. Finally, thanks to a special ultrasound sensor, the recently released Digiline Booklet can easily process fully transparent or semi-transparent labels, which turns into another differentiating element in comparison with a wide variety of traditional thermal transfer printers; and the suitable label stock ranges include, as the company pointed out, PE, PET and PVC film, together with paper. An accidental detachment of booklet labels during the production process is avoided and prevented by «gentle feed-over large guide rollers», tailored to the various label formats available.

Mutagenicity evaluation of 2-amino-4-thiazole acetic acid

R&D

Cristiana Bernini

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9 December 2014

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Mutagenicity evaluation of 2-amino-4-thiazole acetic acid

2-Amino-4-thiazole acetic acid is a building block used in the synthesis of different active pharmaceutical products, specifically in the process of manufacture of Cefotiam. 2-Amino-4-thiazole acetic acid is incorporated in Cefotiam as amide, but it could be re-obtained by hydrolysis, and so it becomes an obvious potential degradation impurity. Following the possibility to have this compound as by-process or degradation impurity, above the identification thresholds outlined by ICHQ3A (1), as foreseen by ICH M7 (2), it needs to be evaluated for its potential genotoxicity.

The Ames Salmonella mutagenicity assays is a short-term bacterial reverse-mutation assay specifically designed to detect mutagenicity in a wide range of chemical substances that can produce genetic damage leading to gene mutation (3).

The presence of a free amine group on an aromatic ring trigger an alert for the potential genotoxicity of this intermediate; a study on this substance was started in order to support the specification now reported by ICH M7

Methods

Test substance 2-amino-4-thiazole acetic acid

2-Amino-4-thiazole acetic acid (CAS 29676-71-9) was obtained from the in-house Chemistry Department, Industriale Chimica and analyzed from the in house Analytical Department. The purity was tested by I.R. spectrum, NMR spectroscopic, and mass spectroscopic analysis.

Experimental procedures

The bacterial reverse mutation assay (Ames test) was performed on five mutant strains of Salmonella typhimurium (TA 1535, TA1537, TA 98, TA 100, TA 102) and Escherichia coli WP2pKM101 according to OECD 471:1997 (4) and GLP.

It is noted that the presumed mutagenic activity was determined by comparing number of revertant colonies in treated plates with those of negative controls; a response is positive if the number of colonies in the treated plates is double or more in respect to control plates.

The enzymatic system for metabolism activation (S9 mix) was prepared adding to S9 (an hepatic homogenate obtained from the liver of adult male rats which had previously been induced with “aroclor 1254” soybean oil solution) to Regensys A and to Regensys B containing respectively phosphate buffered salt solution and glucose-6-phosphate and 153 mg NADP for the activation. S 9 mix was subjected to a sterility control to exclude any possible contamination.

The test substance was tested at the concentration of 50 mg/ml and 7 subsequent dilutions of semi-log intervals equivalent to 15 mg/ml, 4.5 mg/ml, 1.35 mg/ml, 0.405 mg/ml, 0,122 mg/ml 0.037 mg/ml and 0.011 mg/ml. The positive controls were prepared at the concentrations and were used for the strains as reported in Table 1.

Table 1. Positive control for the various strains with and without S9 mix.

Compound	(µg/plate)	Strain	S9
Sodium azide (NaN 3 )	1.5	S.typhimurium TA 1535	without
		S. typhumurium TA 100
9-Aminoacridine (9-AAc)	100	S. typhimurium TA 1537	without
2-Nitrofluorene (2NF)	10	S. typhumurium TA 98	without
Mitomycin C (MMC)	0.5	S. typhimurium TA 102	without
4-Nitroquinoline (4NQ)	50	E. coli WP2pKM101	without
2-Aminoantracene (2AAn)	10	S. typhimurium TA 1537	with
		S. typhimurium TA 98
		S. typhimurium TA 102
		E. coli WP2pKM101
Ciclophosphamide CP)	100	S. typhimurium TA 1535	with
		S. typhimurium TA 100

All positive controls and other reagents were obtained from Sigma-Aldrich (St. Louis, Missoury, USA). All experiments were run with concurrent controls and in triplicate.

Results

Results obtained with different concentrations of 2-ammino-4-thiazole acetic acid in the Ames test are shown in Tables 2-5.

Table 2. Bacterial reverse-mutation test of 2-amino-4-thiazole acetic acid in absence of metabolic activation (trial I). (Revertants/plate).

Conc.	TA 1537	TA 1535	TA 98	TA 100	TA 102	E.coli WP2pKM 101
(mg/plate)	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
50000	15.00±2.65	13.67±2.08	23.00±4.58	114.33±39.31	252.00±31.18	233.33±14.19
15000	12.67±4.04	10,33±1.15	24.33±6.11	137.00±10.54	262.33±11.68	231.67±6.51
0.4500	13.33±1.53	15.00±3.00	23.33±4.04	126.00±35.59	273.67±25.32	232.33±5.69
0.1350	13.33±4.04	15.00±4.58	23.00±3.46	123.67±35.30	250.33±40.51	218.67±12.34
0.0405	16.67±1.53	17.33±2.89	26.67±3.21	116.00±36.66	259.67±33.86	227.33±13.65
0.0122	15.33±2.08	15.33±2.89	24.67±6.51	120.33±3.79	247.33±30.99	220.33±18.50
0.0037	15.00±1.73	13.00±1.73	24.67±6.03	130.67±32.50	267.00±14.73	229.33±11.59
0.0011	14.00±3.00	13.00±2.65	22.33±6.66	104.67±24.58	233.67±24.11	227.00±15.62
NC	13.37±3.51	12.67±1.53	26.33±4.73	112.67±14.57	274.67±22.19	231.33±19.40
PC	599.00±69.20	591.67±127.25	620.33±84.01	1034.33±81.00	1082.67±187.70	1276.33±167.54

NC=controls; PC=positive controls (see Table 1); SD=standard deviation

Table 3. Bacterial reverse-mutation test of 2-amino-4-thiazole acetic acid in absence of metabolic activation (trial II). (Revertants/plate).

Conc.	TA 1537	TA 1535	TA 98	TA 100	TA 102	E.coli WP2pKM 101
(mg/plate)	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
50000	13.33±1.53	14.00±1.00	24.00±5.57	102.67±5.03	268.00±20.07	233.67±6.43
15000	14.33±2.08	14.67±1.15	28.67±1.53	123.67±9.07	241.00±44.24	224.00±15.59
0.4500	15.00±1.00	11.00±3.61	21.00±6.24	113.00±33.41	249.67±9.50	227.33±6.51
0.1350	11.67±1.53	16.00±3.46	24.00±8.19	142.33±28.02	271.67±18.15	213.33±10.12
0.0405	14.33±4.04	15.67±2.31	23.00±6.56	107.00±30.41	257.67±14.47	239.00±2.65
0.0122	13.00±2.00	13.33±4.16	25.00±5.57	121.67±10.26	271.33±24.54	224.33±15.50
0.0037	10.67±1.15	13.00±1.73	23.67±6.43	115.00±10.15	236.33±22.94	238.00±7.81
0.0011	13.67±4.04	15.67±3.06	26.33±4.51	129.00±34.00	242.33±16.65	223.00±2.00
NC	15.67±4.16	13.67±1.53	25.67±7.57	120.00±11.53	251.33±40.80	219.00±14.53
PC	683.00±75.94	650.67±83.03	568.67±65.58	945.67±91.27	1280.67±147.84	1253.67±224.30

NC=controls; PC=positive controls (see Table 1); SD=standard deviation

Table 4. Bacterial reverse-mutation test of 2-amino-4-thiazole acetic acid in presence of metabolic activation (trial I). (Revertants/plate).

Conc.	TA 1537	TA 1535	TA 98	TA 100	TA 102	E.coli WP2pKM 101
(mg/plate)	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
50000	13.67±2.52	14.33±4.16	23.33±2.52	127.67±29.48	247.67±37.74	233.33±9.71
15000	11.00±1.73	16.67±2.31	22.33±6.03	133.00±13.53	250.00±36.35	217.67±17.47
0.4500	13.33±3.79	15.00±4.36	23.67±4.93	122.33±18.45	276.00±9.54	220.33±12.01
0.1350	14.00±4.00	11.33±3.06	24.33±2.52	145.00±17.09	251.00±31.19	217.33±15.14
0.0405	15.67±2.89	14.67±4.04	27.00±4.36	127.67±16.92	258.00±9.00	220.33±4.73
0.0122	11.00±2.65	11.67±2.52	22.67±5.86	114.33±13.32	244.33±15.57	219.00±21.70
0.0037	14.67±3.79	16.33±2.08	19.33±2.08	129.00±30.41	224.33±37.00	216.00±5.57
0.0011	16.00±1.73	14.67±2.52	20.67±4.73	129.67±39.50	272.33±30.66	229.00±21.66
NC	14.67±3.06	15.67±3.51	20.00±4.36	135.00±6.00	254.00±47.62	230.00±10.54
PC	601.33±24.44	531.67±45.28	657.67±31.01	911.67±53.46	1229.67±128.70	1257.67±196.46

NC=controls; PC=positive controls (see Table 1); SD=standard deviation

Table 5. Bacterial reverse-mutation test of 2-amino-thiazole acetic acid in presence of metabolic activation (trial II). (Revertants/plate).

Conc.	TA 1537	TA 1535	TA 98	TA 100	TA 102	E.coli WP2pKM 101
(mg/plate)	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
50000	13.33±1.15	13.00±4.00	21.67±2.08	117.67±30.66	272.33±20.11	224.00±24.52
15000	15.33±4.73	14.00±4.58	25.67±4.51	144.67±23.16	239.67±17.79	235.33±9.87
0.4500	14.33±3.21	14.33±0.58	23.33±2.89	141.00±20.42	244.67±43.19	222.67±15.37
0.1350	16.00±1.73	12.33±0.58	22.33±3.06	139.00±31.51	230.67±21.78	219.33±7.77
0.0405	14.00±4.58	15.33±5.51	26.33±3.21	118.33±33.71	226.67±5.86	236.33±9.07
0.0122	13.00±2.00	14.00±4.00	22.33±2.52	124.33±39.80	244.33±35.44	230.33±0.58
0.0037	14.00±4.36	13.33±3.51	25.33±0.58	108.00±9.17	244.33±40.55	219.67±18.61
0.0011	13.67±1.15	14.67±3.51	25.33±4.62	139.00±27.51	246.00±26.23	216.33±10.12
NC	12.67±3.51	13.67±4.04	25.33±2.31	140.33±20.03	236.67±47.65	221.00±15.87
PC	583.00±61.02	626.33±72.57	578.67±42.34	928.67±137.78	1245.33±188.21	1130.33±81.82

NC=controls; PC=positive controls (see Table 1); SD=standard deviation

Based on the results obtained with different concentrations of 2-amino-4-thiazole acetic acid in the mutagenicity assay, using TA 1537, TA 1535, TA 98, TA 100, TA 102 strains of S. typhymurium, and E. coli KMWP2p, it was observed that 2-amino-4-thiazole acetic acid did not show mutagenic effects , both with and without metabolic activation.

Discussion

We investigated some structural analogue compounds starting from aminothiazole (CAS 96-50-4); the compounds taken into consideration are: 2-amino-4-methylthiazole (CAS 1603-91-4), 2-amino-5-methylthiazole (CAS 7305-71-7), 3-methylisothiazole-5-amine (CAS 24340-76-9) and our compound 2-aminothiazole-4-acetic acid (CAS 29676-71-9).

Aminothiazole was tested in Salmonella typhimurium TA 100 and K.pneumoniae (5). It was not mutagen in S. thyphymurium TA 100; whereas it was mutagen in K. pneumonia. Another set of tests shows that aminothiazole was mutagen only in S.typhimurium TA 98 and TA 1538 (with metabolic activation only), while it was not mutagen in the other S.typhimurium strains (both with and without metabolic activation) (6). Its mutagenic activity was also confirmed by a test in mammalian cells (i.e. L5178Y TK +/- cells); in particular, this test was positive both with and without metabolic activation (6). From these results the mutagenicity of this compound is not clear.

2-Amino-4-methylthiazole was tested in a bacterial reverse mutation assay in accordance with OECD guideline 471 and under GLP conditions. The test was performed on S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102 strains and on E,coli WP2pKM101, both with and without metabolic activation. This compound was clearly non-mutagen in E. coli and S.thyphimurium strains, both with and without metabolic activation (7). Old results show that this compound is mutagen in K. pneumonia and not in S. thyphimurium TA 100 (5). This compound may be considered non mutagen.

2-Amino-5-methylthiazole is a substance which was notified in accordance with the Directive 67/546/CEE and data on its genotoxicity are shown in the database of the registered substances in ECHA website (8). According to these data, the substance was tested in a bacterial reverse mutation assay in accordance with OECD Guideline 471 and under GLP conditions. The test was performed on S. thyphimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, both with and without metabolic activation. 2-Amino-5-methylthiazole was clearly negative in 3 strains of S. thyphimurium, whereas, according to authors, a weak mutagenic response in 2 other genotypically different strains could not be ruled out definitely. No data by in vitro test on mammalian cells or by vivo test were found. These results indicated some mutagenicity for this compound.

3-Methylisothiazol-5-amine was tested in S. thyphimurium and mammalian cells (L5178Y TK +/-). With regard to bacterial assay, the substance was mutagen only in S. thyphimurium TA 98 and TA 1538 (with metabolic activation only), whereas it was not mutagen in the other S. thyphimurium strains (both with and without metabolic activation) (6). Its mutagenic activity was also confirmed by the test in mammalian cells (i.e. L5178Y TK +/- cells), that was positive with and without metabolic activation (9). This compound is considered as mutagen.

Our compound 2-aminothiazol-4-acetic acid, as shown in this paper, is not mutagen in bacterial reverse mutation assay. For a preliminary observation, we can say that the substituent in position 2 of aminothiazole allow to obtain compounds with no mutagenic activity as it is shown by this compound and by 2-amino-4 methylthiazole.

References

International Conference on Harmonisation (2006). Q3A(R2). Impurities in New Drug Substances.
International Conference on Harmonisation (2014). Assessment and control of DNA reactive (Mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk.
Ames BN, Lee FD, Durston WE. (1973). An improved bacterial test system for detection and classification of mutagens and carcinogens. Proc Natl Acad Sci U S A, 70, 782-86.
OECD Guidelines for testing of chemical 471 21^st, July 1997 – Genetic Toxicology: Bacterial Reverse Mutation Assay.
Voogd CE, van der Stel JJ, Verharen, HW. (1983). The capacity of some nitro- and amino-heterocyclic sulfur compounds to induce base-pair substitutions. Mutat Res, 118, 153-65.
Cameron TP, Hughes TJ, Kirby PE, Palmer KA, Fung VA, Dunkel VC. (1985). Mutagenic activity of 5 thiazole compounds in the Salmonella/microsome and mouse lymphoma TK +/- assays. Mutat Res, 155, 17-25.
Radice M, Talamini G, Faccioli F, Coppi G., Mutagenicity evaluation of sulfamethylthiazole and its intermediate 2-amino-4-methylthiazole. Pharma World Magazine 2013; 2:40-5.
ECHA. Registered substances database. 2-Amino-5-methylthiazole (EC n. 423-800-5). Exp. Key Genetic toxicity in vitro. 001.

Authors

Radice M., Fochi C., Industriale Chimica S.r.l., 21047 Saronno (VA), Italy

Faccioli F., Eurofins Biolab S.r.l., 20090 Vimodrone (MI), Italy

Talamini G., Università Cattolica Sacro Cuore, Istituto di chimica agraria e ambientale (PC) Italy

Coppi G., Consultant AFI, Assoc. Farmaceut. Indust., (MI), Italy

A new paradigm for in vitro toxicology tests

Medical Device

Cristiana Bernini

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9 December 2014

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A new paradigm for in vitro toxicology tests

The future may deserve a revolution for in vitro toxicology studies: stems cells might be used to better replicate in vivo real tissues conditions, thus avoiding or at least reducing animal testing. The team led by Massimo Dominici is investigating the technique at the new Mirandola labs

Giuliana Miglierini

The new labs of the Tecnopolo – at the biomedical district of Mirandola, in Italy – will host three different research groups strongly connected one another and fully devoted to improving the collaboration between public institutions – the University of Modena and Reggio Emilia – and private companies in the medtech and pharma sectors in order to revamp competitiveness and innovation: a strategical asset for the biomedical district – one of the main state-of-the-art medtech districts in Europe – that just two years ago was greatly damaged by a very strong earthquake.

Stem cells as biosensors

The Biocompatibility lab, one of the three new labs, will be fully involved in the development of a completely new application for stem cells: under the guide of oncologist Massimo Dominici, director of the Laboratory of Cellular Therapies of the University of Modena and Reggio Emilia and president of the International Society for Cellular Therapy, ISCT, the group will investigate how mesenchymal stem cells might be used in order to in vitro test the toxicity of new or already existing substances. Dominici started to study stem cells applications in the framework of tumoral pathologies in 1997. Years later his attention moved to regenerative medicine, a field were stem cells may be used also as medical devices. “Year after year, we have learnt how to manipulate the cells, how to amplify their number, their behaviour and what they can do”, tells Dominici. The amount of information available today allows the researchers to prefigure a revolutionary use for stem cells, as Dominici tells Pharma World: “We are studying the possibility to use stem cells as sensors able to respond to what is happening inside the cell or in its nearby after exposure to an external substance”. Exposure could generate biochemical and physiological modifications within the cells that might be used in order to evaluate the in vitro toxicology. The new technology under development at the Mirandola’s new lab may represent in future an alternative to classical methods, as for example tests making use of macrophages or fibroblasts. These are immortalised cells, thus not exactly reflecting the biological characteristics of a human cell. “Not differentiated stem cells are very different from fibroblasts – says Dominici – They seldom undergo transformations in vitro and they reflect the tissue from which they were originated”. Stem cells are also very resistant and they often respond to external challenges modifying their functionality without cellular death. This is very important to better understand what is happening within the damaged cell. “Toxicology is often limited by the fact that the substance might kill the cell, thus it is difficult to understand the mechanism at the base of cytotoxicity – tells Dominici. – Stem cells well tolerate antitumoral drugs, for example taxol which is accumulated in the cytoplasm: this may produce may advantages”.

Tissue specific stem cells

A characteristic of stem cells is their ability to differentiate towards different tissues. The group at the Tecnopolo in Mirandola will also use induced pluripotent stem cells (IPS), the Nobel Prize winner technology for medicine in 2012. IPS technology allows to reprogram adult cells, such as fibroblasts, so that they acquire a pluri-potency typical of embryonic stem cells. “Using this technique we are able to obtain a wide set of stem cells, including cells of renal or hepatic origin. These are very important tissues for toxicology, there might be a strong impact with the tested substance”, tells Massimo Dominici.

A new way to test toxicity

The main raw material used by the medtech companies located at the Mirandola biomedical district is bioplastic: is from this that the toxicology tests using stem cells will start, but the method might be easily applied to other materials as well as to existing or new active ingredients to be used by pharma companies. “We can grow the cells in the presence of the substance, observing and measuring the variation of cellular parameters. A main advantage of the method is that stem cells might be used as surrogates of human tissues, thus obtaining not only toxicological information but also data about the impact of the substance on the tissue”, says Dominici.

The goal of the team is to develop new methods to test toxicity as close as possible to cellular reality. A further advantage might be represented by the possibility to reduce animal testing, a target also present in recent official European policies. “Our method might help to limit animal testing just to the critical steps of the development, where is really necessary to use a complex biological system such as a living organism”, comments Dominici.

The new stem cells methodology should undergo validation and regulatory scrutiny prior to become a routine method to test toxicology during drugs and medical devices development. According to what Massimo Dominici told Pharma World, the intention is to built a solid relationship with the regulatory authorities, so to better understand from the beginning how to use the technology in order to provide accurate data to support product development. The researchers at the Mirandola’s lab are currently implementing the operative procedures and setting all the instruments needed to further develop the method. They are planning to start contacts with AIFA, the Italian regulatory agency, as soon as the lab will be fully operative. “We have already contacts with other regulatory authorities, such as the FDA in the United States, and it’s possible that in future we will contact also EMA – says Dominici, who is also president of the International Society for Cellular Therapy. – This represent for me an advantage, as I can have a global overview on the use of stem cells both from the therapeutic and regenerative point of view and from the toxicological one. I wish I can use this advantage to help companies”. The new stem cells’ biosensors should be developed in the form of a kit, so that pharma o medtech companies might directly use it to conduct toxicological evaluation using a ‘read out’ analytical approach.

We shall wait some more time to see the stem cells toxicological method on the market, even if the instrumental equipment needed is already largely available, just need to transfer and implement the new methodology. “Lab free technologies, with no requirement for cellular labelling, are already available. We would like to enter this technological area and supply well characterised stem cells and ancillary reagents. We are going to run tests to give consistency to our approach. At the moment, I like to share the concept at the base of the method, as I think it represents a revolution in screening technologies. I say that as an oncologist. Our method is as near as possible to the biological situation of the human being” comments the director of the new lab.

Public-private synergies

Dominici tells he was positively surprised by the very short time needed for medtech companies in the Mirandola district to built a prototype or to solve complex issues. The new Tecnopolo will host, on a total area of about 800 m², other two labs that will work in close collaboration with the Dominici’s one as well as with the many companies of the biomedical district. “We want to open the labs to companies, that’s why we decided to locate in Mirandola. The new facilities are not entities foreign to the surrounding business environment”, explains Dominici. The entire project borned and has been developed in strict collaboration with several companies already installed in the district, so to avoid the risk to generate a new purely academic start-up, a rare example of public-private collaboration for Italy.

Of the two other labs, the one guided by professor Aldo Tomasi will run validation tests on the stem cells toxicological method using standardised proteomics and chromatographic techniques. The third one is an bioengineering lab directed by professor Luigi Rovati and will be involved in the structural development and prototypisation of the method.

Mirandola’s biomedical district

The biomedical district of Mirandola, at the very core of Emilia-Romagna region in Italy, is one of the main Italian industrial districts and one of the major area of medtech specialisation in Europe. In the district are also located companies in the mechanical engineering and electronic sectors, which are strongly collaborating with the biomedical industry. More than 5 thousand people works directly or indirectly for the medtech industry.

The district borned in 1962 from the idea of producing disposable biomedical articles. In 1965 the first Italian artificial kidney was produced, marking the start of the haemodialysis sector. In 1970 Swiss-based Sandoz was the first multinational company to enter the district thorough the acquisition of Dasco. Bellco, one of the historical companies of the district, was founded in 1972 and still today is a leader in the haemodialysis sector. Following years saw a rapid escalation of the number of companies located in the district. Multinational Baxter and Pfizer joined the district in 1985 and 1986, respectively. In 1990 the consortium Consobiomed brought together the smaller companies of the district; from 1999 the consortium is focusing more on foreign trade. In May 2012, just a month after its 50th anniversary, two earthquakes strongly hit the industrial areas of the biomedical district. Some companies had to move into provisionally new facilities; today the district is again fully operative and the new Tecnolopo represents just the last step of a long history of italian innovation capacity and industrial success.

Chemical Manufacturing Methods for 21st Century. Pharmaceutical Industries

R&D

Cristiana Bernini

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9 December 2014

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Chemical Manufacturing Methods for 21st Century. Pharmaceutical Industries

We asked Professor Nicholas Turner, co-coordinator of CHEM21 for the University of Manchester, to explain the structure and principles of the project

Simone Montonati

An extensive European project dedicated to sustainability in pharmaceutical manufacturing has now reached the mid-point: it goes by the name of CHEM21, the title of which points at the aim of the project: to provide new chemical-transformation methods for the pharmaceutical industry of the 21st century.

“The focus of the project is to develop sustainable methods for the production of pharmaceuticals – Nicholas Turner says. – We essentially designed five work packages (WP): work package 1, essentially defines the problems and try to understand the state of the art of green chemical methods in pharmaceutical production. The WP 2,3 and 4 are about new technologies development: work package two is about metal catalysis but involving non transition metals, package three is about bio-catalysis, package four is on synthetic biology. Work package five is about training methods for future generation scientists in green chemistry. We needed all five work packages, not only about research and development but also understating the problem and designing training packages to train next generation of scientists. The five work packages have to be integrated, also to bring together academic and industrial members to form a very large and extensive consortium, with the required expertise needed to try to solve these problems”.

From this point of view, what is the role of big companies and of SMEs in the project?

“The large companies belong to an organization, the EFPIA – European Federation of Pharmaceutical Industries and Associations – and they define what they would like to see in terms of proposals from the academic community. Their first role is to define the problem in terms of green methods for pharmaceutical manufacturing and then invite people to provide solutions against set targets. The development takes place initially in the academic groups, but when the technology is ready for the testing, they are tested by the pharmaceutical companies in their laboratories. They provide the feedback to improve the design of the methods. The aim is to apply few methods in the companies to manufacture the specific targets. SMEs along the academic groups provide the potential solutions, then they go to the company for testing. The way the financing works is that the EU (by the IMI) funds the academic research and in the SMEs and the companies fund their own research. It is like a public-private partnership. Therefore, there are people working in the academic groups and the SMEs funded by the EU (the IMI) and there are also people deployed by the companies to work alongside them”.

What are in your opinion the most promising aspects of the research about the sustainable Chemistry?

“In our Centre in Manchester our expertise is mainly in biocatalysis and synthetic biology. We have a long history of collaboration with the pharmaceutical industry, but I think in the last five years technology has become more interesting for other members of the chemical industry including energy, fine chemicals, flavors, fragrances, materials. I think it is a very broad technology that can be applied to any chemical industry. It is a new way of producing chemicals: sustainable, low energy, low cost, low environmental impact”.

The funds have been allocated for the project through a public-private collaboration between pharmaceutical companies and the European Union, which co-funds the initiative through the Seventh Framework Programme for Research and Innovation (FP7).

Professor Turner, how do you rate the contribution that the European Commission is providing to the sustainability of chemical processes in terms of general policies and which level has reached Europe compared to the rest of the world, in your opinion?

“I think this is a very high-priority for the European Commission: we had a lot of funding from the EC in this area, not just from IMI, but also from the FP7 and Horizon2020. I think the EU is a major player, probably because the chemical industry is so strong in Europe, and I think that what you are seeing is the result of lobbying by the chemical industry: by BASF, DSM, the farmer companies, the agro-chemical companies. They constitute a significant portion of the manufacturing industry in Europe and I am not sure you appreciate how it is important that this activity remains in Europe and does not go elsewhere. I think what you are seeing is a really sustained activity by the European Community to try and make sure we stay at the leading edge of this kind of research so that our academics and our industrialists are highly competitive globally in sustainable chemistry”.

The size of the project

CHEM 21 brings together the largest public-private consortium in Europe committed to the issue of sustainability of chemical processes. The working group is composed of 23 partners from eight different countries: six pharmaceutical companies, 13 universities and four small or medium enterprises. The project has a couple of leaders formed by the University of Manchester and the English division of Glaxo-SmithKline, and includes, among other participants, Bayer Pharma Deutschland, UK Pfizer, Sanofi Chimie France and the University of Graz, Stuttgart and Leeds. The research, which will last four years and will end in August 2016, will have a total budget of 21.1 million pounds (approximately 26.4 million euro) entirely financed by IMI (Innovative Medicines Initiative). IMI is the largest European public-private initiative dedicated to the development of the more effective and safer drugs; its resources come from members of EFPIA (European Federation of Pharmaceutical Industries and Associations) and the Seventh Framework Programme (FP7 – the EU program dedicated to research and innovation for the period 2007_2013).

The first results

After two years, the project begins to produce the first results. The work package 1, which aims to map the more advanced stages of European pharmaceutical industry and to define the main challenges for 2020, has already produced an impressive work of analysis: the researchers reviewed over 300 documents related to chemical processes, 52 articles dedicated to discoveries in the chemical and pharmaceutical sector and over 6000 projects by members of EFPIA. This survey identified a clear need for stakeholders to push the sustainability of the processes through appropriate measurement tools, effective systems of education and training and a greater reliance on biotechnology.

Instead, the activities of training and education (WP5), started establishing the Young Researchers Network (YRN), created to ease the sharing of experiences, opinions and scientific knowledge between PhD’s and postdocs. The organization, which already includes more than 40 members, does most of its work through a web-community but the participants have the opportunity to meet up at two workshops organized in York (UK) and Graz (Austria). A next meeting, dedicated to the use of biocatalysts, is scheduled for April 2015 in Stuttgart.

Hexagonal spatial grids and nanoscopic blue lights

R&D

Cristiana Bernini

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9 December 2014

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Hexagonal spatial grids and nanoscopic blue lights

The 2014 Nobel Prize for Medicine has awarded the discovery of neuronal mechanisms that transform spatial movements into cerebral reference maps. The Prize for Chemistry has been assigned to the three scientists that developed super-resolved fluorescence microscopy up to a nanoscale resolution

Giuliana Miglierini

Spatial orienteering is a primary capacity of humans as well as all other animals. The sense of sight, one might think, should represent a relevant part of the complex mechanism at the base of the recognition of space inside which we move around and of the building of the consequent mental reference maps. May-Britt and Edvard Moser together with John O’Keefe demonstrated this is not the case, the process occurs entirely at the neurological level without the need of visible point marks, and the results they obtained have been awarded this year with the Nobel Prize for Physiology and Medicine.

Neuronal punctual activation

May-Britt and Edvard Moser are Norwegians working in their lab in Trondheim, which they built in 2007 with a grant of the Kavli Foundation. Far before that, they moved their first steps in neurophysiological research in the lab of John O’Keefe at the University College in London. O’Keefe, now 72, is an American who moved to the UK being fascinated by the British culture and particularly by the BBC, the NHS and Ordnance Survey map, as he told BBC Radio. In his free time, O’Keefe likes to walk in the country side and find his way around and maybe the passion for maps and orienteering suggested him to address its scientific attention towards the study of how the brain build the neuronal map of the space inside which we move. O’Keefe has been awarded with the Nobel Prize for his discovery, in 1971, of the so called place cells, a special kind of neuronal cells located into the hippocampus. Place cell activate and emit neurological signals as rats (but the same applies to all animal as well as to humans) move into the space, a squared box: a different set of activated place cells corresponds to each spatial point the rat pass on moving around, thus indicating that place cells should be somehow involved in the construction of the spatial map for movement. John O’Keefe is now director of the Sainsbury Wellcome Centre for Neural Circuits & Behaviour and professor of Cognitive neurosciences at the UCL University.

Grid cells mapping the space

In 2005 May-Britt and Edvard Moser published in Nature their first results about the role of the socalled grid cells, another type of specialised cells located into the entorhinal cortex of the hippocampus. In the paper, the Mosers described the how grid cells are directionally oriented and how they are activated upon passage of the animal on specific points in space, building a spatial map made up of an ensemble of equilteral triangles. Rats are implanted with cerebral electrodes able to detect a single neuron activation. In such a way, the Mosers recognised a one-to-one correlation between spatial points and grid cell’s activation, ending up in a global map of the spatial movement made of regular hexagons as rats move around in the box. Hexagonal geometry is often used by nature every time there is need for an highly efficient spatial order or energetic stability: examples are the cells of the beehive or the hexagonal geometry of benzene and other aromatic compounds, even at the nanolevel as graphene. The Mosers’ hypothesis is that the brain uses an hexagonal code to represent and transfer spatial information into neurological networks. Hexagonal reference schemes are persistent also in the dark and are apparently somehow linked and oriented by ‘connections’ with the box’s borders. Grid cells are stratified within the entorhinal cortex, from up to bottom, according to the size of the hexagonal grid they generate.

As rats move around, each grid cell is specifically activated and generates an electric signal in correspondence to a single point in space. The resulting code, summing all different activations and thus mapping the space, is then transmitted to the hippocampus, which is responsible for the associations between space, time and action that are at the base of thoughts and memory. Grid cells functionality and behaviour might be involved in early stage neuronal pathologies such as Alzheimer, where loss of the capability of spatial orienteering is often an early symptom, suggest Mosers’ results. The Nobel winning couple is now planning to further investigate how spatial recognition mechanisms develop from birth on in order to adapt to the external environment.

Nanoscale optical microscopy

A wall has been torned apart by the three winners of the 2014 Nobel Prize for Chemistry: thanks to the innovations the introduced, optical microscopy is no longer limited by the Abbe’s limit (Box “Limits for Optical Microscopy”), corresponding approximately to half the wavelength of the light used by the microscope. Traditional optical microscopy has a resolution up to approx. 200 nm, thus it is useful to detect bacteria but not viruses, proteins or small molecules. The new techniques introduced by Stefan Hell, director of the Max Planck Institute for Biophysical Chemistry in Gottingen, William E. Moerner, physical chemist from Stanford University and Eric Betzig, physics at the Howard Hughes Medical Institute, allow now to reach resolutions up to nanometers, and ‘nanoscopy’ might become a more convenient term to identify a technique that today may detect single molecules without the disadvantages typical of electronic microscopy (i.e. the need of high vacuum which prevent the use of living cells as samples).

Stefan Hell introduced in 2000 the Stimulated Emission Depletion (Sted) method of analysis: a first laser beam enlights the fluorescently marked sample; a second laser is used to ‘remove’ fluorescence, like an eraser, except from a nanometer-wide portion of the sample which is thus magnified. The global image of the sample is reconstructed upon acquisition and addition of many single images at the nano-scale level, thus allowing to overcome Abbe’s limit.

Blue light for single molecule detection

Betzig and Moerner further developed the method and separately introduced Single fluorphore microscopy (SFP), a technique where a small quantity of a fluorescent protein (i.e. GFP, green fluorescent protein) is added to the analytical sample. Using a very weak blue light the protein become temporarily fluorescent: the repeated enlighting of the sample allows for the detection, at each passage, of a different subset of fluorescent proteins. The global image of the sample is obtained summing the single frames. Using the technique of GFP’s dispersion into gel, Moerner has been the first scientist able to detect a single molecule using optical microscopy. Eric Betzig has applied the technique to the study of specific components within the cells.

Antoni van Leeuwenhoek invented optical microscopy in the second half of 1600 as a method to better test carpet’s quality; he was the first to observe red blood cells, somatic muscle cells and protozoans. After 400 years, a new era is opening for optical microscopy thanks to Nobel Prize’s winning innovation.

LIMITS FOR OPTICAL MICROSCOPY

Ernst Abbe in 1873 established the optical limit for the resolution of two different points in a sample in half of the wavelength of the radiating light, corresponding to approximately 200 nm.

A traditional optical microscope can be used to detect animal cells (50 m) and, with some more effort, bacteria (500 nm). Viruses (100 nm), proteins (10 nm) and small molecules (few nm) are not detectable this way.

Very small microorganisms or molecules are detectable with electronic microscopy with resolutions up to 100 pm. Yet, this technique too has a limit: it requires high vacuum conditions, thus it is not compatible with in vivo cell analysis.

Cristiana Bernini

Mirandola’s biomedical district

The size of the project

The first results

LIMITS FOR OPTICAL MICROSCOPY

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