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novel Schiff base metal complexes

Posted in Bacterial Resistance, Curation, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Infectious Disease & New Antibiotic Targets, Microbiology, tagged antimicrobial activity, IR, Photo fluorescence, Schiff base, UV on November 28, 2015| Leave a Comment »

novel Schiff base metal complexes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Synthesis and Characterisation of some novel Schiff base metal complexes: Spectral, XRD, Photoluminescence and Antimicrobial Studies
Rajendran Jayalakshmi and Rangappan Rajavel* – Department of chemistry, Periyar University, Salem-636 011, Tamil Nadu, India

Chem Sci Rev Lett 2015; 4(15); 851-859 Article CS072046073 http://chesci.com/articles/csrl/v4i15/11_CS072046073.pdf

Schiff base ligands are potentially capable of forming stable complexes with most transition metal ions, which act as model compounds for biologically important species. Cu (II), Co (II), Ni (II) and Mn (II) Schiff base metal complexes was prepared by the condensation of benzil and 2,6-diamino pyridine in 1:1 molar ratio. The chemical structures of the Schiff base ligand and its metal complexes were confirmed by various spectroscopic studies like IR, UV-VIS, 1H NMR, TGA/DTA, Powder XRD, Molar conductance, and Photo luminescence. The free Schiff base ligand and its complexes have been tested for their antibacterial activity using disc diffusion method. From the biological studies, the complexes exhibit more activity than the ligand.

Metal-chelate Schiff base complexes have continued to play the role of one of the most important stereo chemical models in main group and transition metal coordination chemistry due to their preparative accessibility, diversity and structural variability(Garnovskii.,1993). Schiff bases have gained importance because of physiological and pharmacological activities associated with them. Schiff base metal complexes have ability to reversibly bind oxygen in epoxidation reactions [1], biological activity [2-4], catalytic activity in hydrogenation of olefins [5, 6] non-linear optical materials and [7] and photochromic properties [8]. The Schiff base complexes were also used as drugs and they possess a wide variety of antimicrobial activity against bacteria, fungi and it also inhibits the growth of certain type of tumours [9, 10]. The symmetric nature of a number of homo dinuclear transition metal derived metallobiosites and of the ability of the individual metal ions to have quite distinct roles in the functioning of the metalloenzyme concerned has led to a search for symmetrical dinucleating ligands which will give binuclear complexes capable of acting as models for the metallobiosites [11, 12]. 2, 6-Diamino-pyridine is a mediumproduction-volume chemical used as a pharmaceutical intermediate and a hair dye coupler in oxidation/permanent formulations. Although mutagenic activity has been reported, here in the present work we report the formation of Schiff base ligand from the condensation of 2, 6-diamino pyridine with benzil and the complexation with metal ions to form potentially active macrocyclic binuclear Schiff base metal complexes.

Synthesis of Ligand ((6E)-N2-((E)-2-(6-aminopyridin-2-ylimino)-1, 2-diphenylethyidine) pyridine-2, 6-diamine) The Schiff base ligand ((6E)-N2-((E)-2-(6-aminopyridin-2-ylimino)-1,2-diphenylethylidine)pyridine-2,6-diamine) were prepared by the drop wise addition of a solution of 2,6-diaminopyridine (0.22 g, 2 mmol) in ethanol (20 ml) to a stirred solution of benzil (0. 21 g, 1 mmol) in ethanol (20 ml). After the addition was completed, the mixture was condensed for 3 h at 900C. A brown precipitate solution was formed. The solution was kept for slow evaporation. The formed brown precipitate was filtered and washed with ethanol and then dried in air. Yield: 0.44 g (53%). Anal Calcd. For C24H20N6: C-73.45, H-5.14, and N-21.41. Found: C-73.38, H- 5.09, and N-21.40.

Synthesis of Schiff base metal complexes ([M2 (L) 2].4(OAc)) The macrocyclic binuclear Schiff base metal complexes (Cu(II), Ni(II), Co(II) and Mn(II)) were prepared by the condensation of 20 ml DMF solution of synthesized ligand (2mmol) adding to the constant stirring of 20 ml of ethanolic solution of metal salt (2 mmol M (where M = Cu2+, Ni2+, Co2+, Mn2+)) which was boiled for 3 hour under reflux. The coloured solution was formed. It was kept for slow evaporation and then collected the precipitate. It was filtered and washed with ethanol and then dried in vacuum.

The resultant macro cyclic Schiff metal complexes were colored powders, and stable for a long time in the open atmosphere. The analytical data and some of the physical properties of the Schiff base ligands and their binuclear metal complexes were summarized. All the metal complexes were sparingly soluble in general organic solvents, and soluble in DMF and DMSO, but insoluble in H2O, EtOH and MeOH. From the molar conductivity data, we clearly found the metal complexes were electrolytic in nature. The structural studies of the ligand and their complexes were done by spectroscopic methods.

Molar conductance The molar conductivity measurements commonly employed in the determination of the geometrical structure of inorganic compounds at infinite dilution. The molar conductance of binuclear Schiff base complexes was dissolved in DMSO and recorded (10-3M molar conductivity solution) at room temperature (Table 1). The complexes showed the range of molar conductance (127-134 ohm1 cm2mol1 ). From these values, we concluded that the complexes were electrolytic in nature. From the molar conductance, we concluded that the anions were outside the coordination sphere and not bonded to the metal ion therefore, these complexes may be formulated as [M2L2]4Z where, Z = acetate ion.

Table 1 Molar conductance and magnetic moment data of Schiff base binuclear metal complexes

S.No Compounds Solvent Molar conductance Type of electrolyte Magnetic moment Geometry
Λm (ohm1 cm2 mol-1 ) μ eff B.M

1. [Cu2(L)2]4(OCOCH3) DMSO 132 1 :2 electrolyte 1.74
2. [CO2(L)2]4(OCOCH3) DMSO 127 1 : 2 electrolyte 4.83
3. [Ni2(L)2]4(OCOCH3) DMSO 134 1 : 2 electrolyte 2.91
4. [Mn2(L)2]4(OCOCH3) DMSO 129 1 : 2 electrolyte 5.82 Octahedral

IR Spectra of the free ligand and their binuclear metal complexes Vibrational spectra provide valuable information regarding the nature of functional group attached to the metal ion in the complexes. The IR spectra of the complexes show very similar spectra to one another. These spectra indicates the replacement of Ѵ(NH2) and Ѵ(C=O) of the starting materials with Ѵ(C=N) which suggest the occurs of the condensation reaction between amine and carbonyl groups [13]. Selected vibration bands of ligands and their metal complexes are given in Table 2. From the IR spectral analysis, the assignment of the important bands was made and recorded. In order to give a conclusive idea about the structure of the metal complexes, the IR bands of metal complexes were compared with free Schiff base ligand. The appearance of a strong, broad band at 3177 cm−1 in the spectra of the free ligands was assigned to ν (NH2). The IR band was shifted in the region (3063–3198 cm -1 ) shows the involvement of primary amine nitrogen atom coordinate to the metal ion for all the Schiff base metal complexes (Ray et al., 2009) after the complexation. The appearance of the band at 1629 cm -1 which may be assigned to the azomethine group Ѵ(C=N) vibration, indicate the condensation of the amino group of 2,6-diamino pyridine with the carbonyl group of benzil and formation of the proposed Schiff base. The IR spectra of all metal complexes show significant changes compared to free Schiff base ligand. After Complexation, the positions of the Ѵ(C=N) were shifted in the range (1660-1667cm1 ) indicates the participation of the azomethine group in complex formation (Singh et al., 2010). The position of an N – atom of the azomethine group and group of the pyridine ring in coordination is further supported by the presence of new bands in the range from 470–495cm-1which is assignable to (M-N) vibration. From the spectroscopic behaviour of metal complexes of pyridine, after the complexation the ring deformation found at 797 cm-1 and 711 cm−1. It was clearly indicate that the free pyridine is shifted to higher frequencies [14], and the coordination takes place via the pyridine nitrogen, as previously reported for pyridine complexes [15]. Therefore, this shift is clearly indicates the participation of pyridine in complex formation. The appearance of band range from 1660 cm−1 to 1438 cm−1 were due to symmetric stretching frequency and asymmetric stretching frequency of acetate ion. This clearly indicates that the acetate ions were coordinated outside of the coordination sphere.

Table 2 IR spectral data (cm-1 ) of the Schiff base (L) and their binuclear metal complexes

1H NMR spectrum of ligand and its macro cyclic binuclear metal complexes The 1H NMR data of the Schiff base (L) and the metal complexes were recorded in DMSO-d6 (Table 3). Assignment of 1H NMR signals were made according to their reported results for 2,6-diaminopyridine and its complexes [16-20]. The 1H-NMR spectra of ligand and its metal complexes show different peaks in the range 6.99-7.94 ppm corresponding to H3, H4, and H5 protons indicate unsymmetrical binding of the ligand to M (II) complexes. In the 1H NMR spectrum of M (II) complexes, singlet signal of the pyridine-NH2 (s, 3.1 to 3.7 ppm) and multiplet signals of aromatic protons (m, 6.72 to 7.94 ppm) of Schiff base (L) shifted compared to the starting material which suggests coordination through nitrogen atom of the azomethine group. For the metal complexes, a single sharp signal is appeared (region from 2.1 – 2.6 ppm) in the 1H NMR spectrum, suggest that the acetate ion is present in the outside coordination sphere of the metal complexes.

Table 3 1H NMR spectrum of ligand and its macro cyclic binuclear metal complexes

Electronic absorption spectra and magnetic moment measurements The electronic spectrum of the Schiff base ligand in DMSO (Table 4), the absorption band observed at 274 nm were assigned to π→π* transition and the band at 386 nm were assigned due to n→π* transition associated with the azomethine chromophore (-C=N).The absorption bands of the complexes are shift to longer wave numbers compared to that of the ligand [21]. For [M2 (L) 2]4(OCOCH3) complexes, the electronic absorption band occurs at 468-474 nm due to charge transfer from ligand to metal ion (LMCT). The obtained Cu (II) complexes exhibits a band at 652 nm assigned to 2Eg → 2T2g transition which is in conformity with octahedral geometry around the Cu (II) ion (Patil et al., 2010; Lever, 1968). The obtained magnetic moment value (µeff) for Cu(II)complex is 1.89 BM indicating that magnetic exchange occurs between the two copper sites and also supports octahedral geometry of Cu(II) complex [22]. The electronic absorption spectra of Co (II) complexes showed a band at 648 nm corresponding to 4T1g(F)→4A2g(F) transition and also the obtained magnetic moment value is 4.84 BM which confirm the octahedral geometry of the complex [23]. For the Ni (II) complex, it has the 3.06 BM magnetic moment value and the electronic spectrum showed a band at 645nm corresponding to 3A2g (F) →3T1g (F) transition which is consistent with the octahedral geometry of the complex. The Mn (II) binuclear complex shows bands at 633 nm corresponds to 6A1g→4T2g (4G) transitions and 5.82 BM magnetic value were compatible to an octahedral geometry of the ligand around manganese (II) ion [24].

Table 4 Electronic Spectral data of Schiff base ligand and their macro cyclic binuclear metal complexes

Compounds Electronic absorption spectra (nm) Magnetic moment Geometry of
π→π* n→π* L→M d-d value(µeff)BM the complex
(nm) (nm) (nm) (nm)
C12H12N6 (ligand) 274 342 – – –
[Cu2(L)2]4(OCOCH3) 268 340 474 652 1.89
[CO2(L)2]4(OCOCH3) 266 338 468 648 4.84
[Ni2(L)2]4(OCOCH3) 256 339 471 645 3.06 Octahedral
[Mn2(L)2]4(OCOCH3) 261 341 474 636 5.82

TGA and DTA studies By using TGA and DTA analysis the thermal stability of the complexes were explained. The observation thermogram and curves (Table 5, Figure 2) were obtained at a heating rate of 100C/min over a temperature range of 40–7300C. The complex was stable up to 1600C and its decomposition started at this temperature. In the thermal decomposition process of the Cu (II) complex proceeds two steps of the mass losses corresponded to acetate, and NH2 leaving in the first, and second stages of the decomposition. The decomposition of the Cu (II) are irreversible. The Cobalt complexes were stable up to 2000C and its decomposition started at this temperature. The Cobalt (II) complex was decomposed in two steps with the temperature ranges from 200-470˚C corresponding to the loss of acetate and NH2 respectively. The Ni (II) complexes were stable up to 180oC and its decomposition started at this temperature. In the decomposition process of the Ni(II) complex, the estimated mass loss of the first step 6.85(6.91) corresponded to the loss of four acetate group and the second stage the liberation of four NH2 unit respectively, shown in table 6. The decomposition of the Nickel complex was irreversible. The thermal decomposition of the Manganese complex was stable up to 1700C and its decomposition started at this temperature. Thereafter, they start the decomposition process of the Mn (II) complex and weight loss observed in the temperature range 170-4600C, the mass loses corresponded to four acetate and four NH2 leaving in the first and second stages of the decomposition. The decomposition of the Mn (II) complexes are irreversible. The amount of acetate and NH2 groups stoichiometrically corresponding to the weight losses are given in the proposal chemical formulas of complexes.

Powder XRD Analysis Synthesized Schiff base metal (II) complexes were subjected to Powder X-ray diffractograms in the range (2ɵ = 10– 600 ) were shown in (Figure 3). Among the metal complexes Ni (II) complex shows well defined crystalline sharp peak which indicate the sample were crystalline nature. The appearance of crystallinity in the Schiff base metal complexes is due to the inherent crystalline nature of the metallic compounds. The average grain size (dXRD) of the Ni (II) complex is 32 nm which was calculated by using Scherer’s formula (Dhanaraj and Nair, 2009a,b) suggesting that the Ni(II) complex are nanocrystalline.

Table 5 Thermo gravimetric data of metal complexes

Figure 2 DTA/TGA Curve for metal complexes

Figure 3 Powder X-ray diffractogram for Ni complex

Fluorescence spectra The Schiff base and its binuclear metal complexes were analysed by the photoluminescence emission spectra (Figure 4) and recorded in DMSO at room temperature. Comparing with Schiff base ligand and its macro cyclic binuclear metal complexes, the metal complexes have strong fluorescence intensity than Schiff base. Among the metal complexes the Co (II) complex exhibited a strong fluorescence emission at 400 nm (Flourescence intensity 713) with excitation at 269 nm. The quenching of metal (II) complexes indicates that the ligand has a less potential photo active than metal (II) complexes.

Figure 4 Fluorescence spectra for all metal complexes

In vitro antimicrobial activity of Schiff base ligand and their metal complexes By using broth micro dilution procedures, the Schiff base ligand and their metal complexes were screened separately against for two Gram positive bacteria (Staphylococcus aureus and B. Subtilis), two Gram negative bacteria (E. Coli and S. typhi) and the fungi (A. fumigatus) for their antimicrobial activity. When the activity of Schiff base ligand and their metal complexes were increased by increasing the antimicrobial screening concentration (Table 6, Figure 5), because the concentration plays an important role in the zone of inhibition and the chelated metal complexes deactivate the various cellular enzyme [25]. Metal complexes show considerable antimicrobial activity even at low concentration and also more toxicity towards Gram-positive strains, Gram-negative strains and fungi compare with Schiff base ligand. The antimicrobial data shows that the copper complex noticed an excellent activity against bacteria and fungi than other metal complexes. The different antimicrobial activity of different metal complexes depends on the impermeability of the cell or the difference in ribosomes in microbial cell (Sengupta et al., 1998).

Table 6 Antimicrobial activity of Schiff base ligand and their metal complexes

Figure 5 Anti-Bacterial Activities of the Schiff Base and Its Binuclear Metal Complexes against Gram positive and negative Bacterias

Conclusion Macrocyclic binuclear metal (II) complexes was synthesized by using condensation method of a novel Schiff base ligand derived from 2, 6-diamino pyridine and benzil. The data which have been the physico chemical and spectral studies provides excellent structure and chemical composition of Schiff base and its metal complexes. The electronic absorption spectra, IR spectra and magnetic moment value reveals that the metal complexes were octahedral geometry and the Schiff base coordinated through six nitrogen atoms of azomethine group and pyridine ring. Powder XRD data reveals that the Ni(II) complex was nano crystalline structure. Based on the photo luminescence studies, we have confirmed the metal complexes were more potential photo active than Schiff base. The in vitro antimicrobial studies of metal (II) complexes showed better activity than Schiff base.

References

[1] Viswanathamurthi P, Natarajan K, Synth.React.Inorg.Met.-Org.Chem 2006; 36:415-418.

[2] Ren S, Wang R, Komastu K, Krause P.B, Zyrianov Y, Mckenna C. E, Csipke C, TokesZ. A, Lien E. J, J. Med. Chem 2002; 45: 410

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Global Clinical Trials For Brain Ischemia 2015 by Market Research Store – Medgadget.com (blog)

Posted in Uncategorized on November 28, 2015| Leave a Comment »

“Brain Ischemia Global 2015 Clinical Trials Review, H2″ provides an detailed overview of Brain Ischemia scenario. Report includes top line data relating on

Sourced through Scoop.it from: www.medgadget.com

See on Scoop.it – Cardiovascular and vascular imaging

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Hospital Acquired Infections

Posted in Curation, Microbiology, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Population Health Management, tagged Antibiotic resistance, hospital acquired infection on November 28, 2015| Leave a Comment »

Hospital Acquired Infections

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Hospital infection control in the era of superbug outbreaks
By Leslie Small

http://www.fiercehealthcare.com/special-reports/hospital-infection-control-era-superbug-outbreaks-special-report

Long before superbug outbreaks tied to a specialized medical scope sickened and killed patients across the country, infection control has been a major priority for hospitals.

But the need to curb infections has become even more pressing now that the shift from a fee-for-service model to value-based payments has led the federal government to increasingly tie reimbursements to patient outcomes. An industry trend toward transparency also has made it easier for the public to see the danger of a hospital stay.

One in 25 hospital patients acquires at least one healthcare-associated infection (HAI), according to data from the Centers for Disease Control and Prevention. There were an estimated 722,000 HAIs in U.S acute care hospitals in 2011, about 75,000 hospital patients with HAIs died during their hospitalizations, and more than half of all HAIs occurred outside of the intensive care unit.

The problem has gained more attention in recent months, when reports surfaced of patients contracting a particularly lethal antibiotic-resistant superbug, carbapenem-resistant Enterobacteriaceae (CRE), after undergoing procedures that involved a device known as a duodenoscope. A Food and Drug Administration advisory panel concluded this month what hospitals involved in the outbreak already knew–that the ability of CRE and other dangerous pathogens to stay on scopes after cleaning puts patients at a significant risk.

But while the problems posed by such outbreaks are clear, finding solutions to them–particularly in a seldom-static healthcare industry–is anything but. To help chart a way forward, this special report from FierceHealthcare examines advice from experts and hospital leaders who have learned valuable lessons from the front lines of hospital infection control.

Rise of a superbug jeopardizes patient safety

Hospital infection control in the era of superbug outbreaks
By Leslie Small

http://www.fiercehealthcare.com/special-reports/rise-superbug-jeopardizes-patient-safety

The recent carbapenem-resistant Enterobacteriaceae (CRE) outbreak burst onto the scene when reports surfaced in February that it caused two patient deaths at UCLA’s Ronald Reagan Medical Center. In addition to the California outbreak–which also sickened patients at Cedars-Sinai Medical Center in Los Angeles–cases of the superbug linked to duodenoscopes also cropped up at hospitals in North Carolina, Pittsburgh, Chicago and Seattle.

The Centers for Disease Control and Prevention (CDC) has cautioned hospitals that they must do more to mitigate the threat of CRE, which it dubbed “nightmare bacteria” due to their resistance to even last-resort antibiotics. When it reaches the bloodstream, CRE can kill up to half of all patients it infects.

The Food and Drug Administration (FDA) issued a warning in February that the complex design of duodenoscopes makes them difficult to sterilize even when hospitals follow the device manufacturers’ instructions. However, the FDA has refused to take the devices off the market because they are used for the potentially life-saving procedure known as endoscopic retrograde cholangiopancreatography (ERCP), a technique that diagnoses and treats cancers and other digestive diseases. The agency estimates that 500,000 ERCPs are performed each year. A special advisory panel recently endorsed this decision, though it urged the FDA to better protect patients from the infection risk posed by duodenoscopes,FierceHealthcare has reported.

The panel was also critical of major duodenoscope manufacturer Olympus, which declined to participate in the advisory panel’s forum but says it has supplied the FDA with data to prove that its updated cleaning instructions and new cleaning brush allow for safe reprocessing.

Indeed, news surfaced recently that the company was aware of the infection risk associated with the devices in 2013, which it communicated to European hospitals two years before the UCLA outbreak. The situation has led California lawmaker, Rep. Ted Lieu (D-Los Angeles) to call for congressional hearings into the matter. Meanwhile, Olympus faces two patient-driven lawsuits, and Virginia Mason Hospital in Seattle also has pursued legal action against the manufacturer.

The outbreaks have left many hospitals wondering what to do to make sure patients are safe and still have access to important medical devices. For its part, the FDA panel did not outright endorse any specific sterilization method.

Jackie Caynon, pictured right, a lawyer with more than 18 years of health law experience, and partner and co-chair of Mirick O’Connell’s Health Law Group, told FierceHealthcare in an exclusive interview that the answer has to come from each hospital’s unique risk management assessment.

“I’ve heard some hospitals say, you know these things are really life-saving, so if we get rid of the product we won’t be able to, obviously, save lives,” he said. But for others, he said, the risk may be too great.

“To me it just seems too risky to use it,” Caynon said. “If you’re going to do informed consent, I could see you saying to the patient ‘oh you know, this could save your life, but we won’t know until we actually go in there and look, but you run the risk of having a CRE infection because we cannot guarantee that we can properly clean this device.'”

And now that the infection risk surrounding the devices has been made public, “I think you’re going to have a lot of patients that are going to say ‘I don’t want you to use that device, period,'” he said.

Regardless of what each individual facility decides to do about the scopes, it would be a mistake to hold hospitals responsible for manufacturers’ mistakes or regulatory failures, according to Caynon.

“Holding hospitals and physicians liable here is kind of going after the wrong folks,” he said, because “the hospital is just as much of a customer as patients (are).”

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anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV)

Posted in Frontiers in Cardiology and Cardiovascular Disorders, Image Processing/Computing, Immunodiagnostics, Innovation in Immunology Diagnostics, Innovations, Medical Imaging Technology, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, tagged ANCA, PET on November 28, 2015| Leave a Comment »

anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV)

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Positron Emission Tomography scanning in Anti-Neutrophil Cytoplasmic Antibodies-Associated Vasculitis

Kemna, Michael J. BSc; Vandergheynst, Frédéric MD; Vöö, Stefan MD, PhD; Blocklet, et al.

Medicine: May 2015;94(20) – p e747 http;//dx.doi.org:/10.1097/MD.0000000000000747

Tools for evaluation of disease activity in patients with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) include scoring clinical manifestations, determination of biochemical parameters of inflammation, and obtaining tissue biopsies. These tools, however, are sometimes inconclusive. 2-deoxy-2-[¹⁸F]-fluoro-D-glucose (FDG) positron emission tomography (PET) scans are commonly used to detect inflammatory or malignant lesions. Our objective is to explore the ability of PET scanning to assess the extent of disease activity in patients with AAV.

Consecutive PET scans made between December 2006 and March 2014 in Maastricht (MUMC) and between July 2008 and June 2013 in Brussels (EUH) to assess disease activity in patients with AAV were retrospectively included. Scans were re-examined and quantitatively scored using maximum standard uptake values (SUVmax). PET findings were compared with C-reactive protein (CRP) and ANCA positivity at the time of scanning.

Forty-four scans were performed in 33 patients during a period of suspected active disease. All but 2 scans showed PET-positive sites, most commonly the nasopharynx (n = 22) and the lung (n = 22). Forty-one clinically occult lesions were found, including the thyroid gland (n = 4 patients), aorta (n = 8), and bone marrow (n = 7). The amount of hotspots, but not the highest observed SUVmax value, was higher if CRP levels were elevated. Seventeen follow-up scans were made in 13 patients and showed decreased SUVmax values.

FDG PET scans in AAV patients with active disease show positive findings in multiple sites of the body even when biochemical parameters are inconclusive, including sites clinically unsuspected and difficult to assess otherwise.

Granulomatosis with polyangiitis (GPA; Wegener’s) is an inflammatory disease entity affecting small to medium vessels. It is, together with microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA; Churg Strauss Syndrome), characterized by the presence of anti-neutrophil cytoplasmic antibodies (ANCA) and they are frequently grouped together under the term ANCA-associated vasculitis (AAV).¹

Early diagnosis and assessment of the extent of disease activity are important for adequate therapeutic decisions.¹ Multiple tools may be helpful, such as biochemical parameters of inflammation, imaging techniques, and tissue biopsies. Even though these tools suffice to diagnose active disease in most episodes, the results can sometimes be inconclusive. In particular, it is sometimes problematic to determine whether symptoms are due to active disease, vasculitic damage, and/or treatment-related side-effects.

2-deoxy-2-[¹⁸F]-fluoro-D-glucose (FDG) positron emission tomography (PET) scanning is used for detecting high glucose metabolism in malignancies, infectious, and auto-immune diseases.^2–4 Co-registration with computed tomography (CT) allows the increased FDG uptake to be localized to the underlying anatomy. PET scanning has been proven to be a useful diagnostic tool in large vessel vasculitis.^5–8 PET scanning can visualize glucose-consuming inflamed vessels, provided that their diameter is >4 mm. The limited spatial resolution was previously thought to be insufficient to detect the involvement of small- and medium-size vessels.^6,7 Recent studies, however, have shown that PET scans show abnormalities in patients with ANCA-associated vasculitis.^9–11 This novel imaging technique may therefore be a useful tool for diagnosing active disease and, in addition, to assess the severity and the extent of the disease. The latter may be relevant to detect occult diagnostic biopsy sites as previously demonstrated in sarcoidosis.¹²

The objective of our study is to explore the ability of PET scanning to assess the extent of disease activity in patients with AAV.

Study Population

Consecutive PET scans were performed in patients with AAV at Maastricht University Medical Center (MUMC) between December 2006 and March 2014 and at Erasme University Hospital (EUH) in Brussels between July 2008 and June 2013 and were retrospectively included. All patients fulfilled a diagnosis of GPA according to the 2012 revised International Chapel Hill Consensus Conference Nomenclature.¹³ Patients were previously treated according to the recommendations of the European League Against Rheumatism (EULAR).¹⁴ Disease states were defined according to the EULAR recommendations.¹⁵ A PET scan was performed in patients with clinically suspected disease activity (diagnosis or relapse), whereas other tools for evaluation of activity were inconclusive. The possibility of an active bacterial or viral infection was excluded by culture, serology, and persistence of symptoms despite empirical antibiotic treatment. This study was carried out in compliance with the Helsinki Declaration.

Diagnostic Parameters

An extensive diagnostic work-up was done in all cases, including analysis of clinical features, laboratory assessment, imaging techniques, and, if appropriate, a biopsy. Laboratory assessment included high-sensitivity C-reactive protein (CRP, cutoff value ≥10 ng/mL) levels, ANCA levels, and urine analysis at the time of scanning. Hematuria was defined as ≥10 erythrocytes in a urinary sediment, combined with dysmorphic erythrocytes and/or red blood cell casts. In Maastricht, ANCA levels were determined using the Fluorescent-Enzyme Immuno-Assay (FEIA) method.¹⁶ FEIA detection for both proteinase-3 (PR3) and myeloperoxidase (MPO) antibodies were fully automated as performed in a UniCAP 100 (Pharmacia Diagnostics). Values ≥10 AU were considered positive.

A whole-body [¹⁸F]-FDG-PET/CT scan was performed in both centers. In Maastricht, a Gemini_ PET-CT (Philips Medical Systems) scanner with time-of-flight (TOF) capability was used, together with a 64-slice Brilliance CT scanner. This scanner has a transverse and axial Field of View (FOV) of 57.6 and 18 cm, respectively. The spatial resolution is around 5 mm. In Brussels, a Gemini_ PET-CT (Philips Medical Systems) scanner was used without TOF capability, but with the same PET FOV and spatial resolution, together with a 16-slice Brilliance CT scanner.

Patient Characteristics

Thirty-three patients were included; an overview of the patient characteristics is shown in Table 1. Twenty patients were positive for PR3-ANCA at diagnosis, 9 patients for MPO-ANCA, and 4 patients were ANCA-negative.

Table 1
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Forty-four PET scans were made during an episode of suspected disease activity (Table 2). Eleven scans were performed at diagnosis and 33 scans at a suspected relapse. The suspected relapses occurred after a median of 68 (30–113) months since diagnosis. In 5 patients, ≥2 consecutive episodes occurred during which a PET scan was performed. These patients were in remission between episodes.

Table 2
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http://images.journals.lww.com/md-journal/LargeThumb.00005792-201505040-00006.TT2.jpeg

Results of PET Scans During Suspected Disease Activity

All PET scans during an episode of suspected disease activity except 2 revealed enhanced non-physiological FDG uptake. Table 3 shows the anatomic location of the positive sites and the corresponding median SUVmax values. The majority of these sites disclosed a SUVmax value between >2.5 and <6. Examples of PET/CT images of patients with AAV are shown in Figures 1 and Figures 2.

Table 3
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http://images.journals.lww.com/md-journal/LargeThumb.00005792-201505040-00006.TT3.jpeg

In our study, PET scans in AAV patients revealed positive findings in multiple sites of the body, including sites not clinically suspected and difficult to assess otherwise. PET scans may show FDG-positive findings during episodes in which other tools for evaluation of disease activity are inconclusive.

Similar to our findings using Gallium-67 [⁶⁷Ga] scintigraphy¹⁷ in patients with GPA, PET scans seem to be a sensitive tool to assess disease activity. In our current study, all but 2 scans showed non-physiological FDG uptake during an episode of clinically suspected disease activity. Compared with gallium scanning, however, PET scanning offers additional information. First, Gallium scintigraphy suffers from practical limitations, such as the required interval between time of injection of the radiopharmaceuticals and time of scanning (48–72 hours) and the high radiation exposure. Second, the spatial resolution is higher in PET scans. Third, a low-dose CT scan may be used concomitantly to correlate the FDG uptake with the precise anatomical location. In sarcoidosis, PET scans are of value in detecting occult diagnostic biopsy sites.¹² In our cohort, 41 clinically occult sites were found on the PET scan, and in 1 patient this resulted in a diagnostic biopsy.⁹

Whether hotspots on the PET scan can be attributed to activity of vasculitis is sometimes difficult to assess. A biopsy of PET-positive lesions would result in a definitive diagnosis. However, such a strategy is not realistic, as it does not correspond to routine clinical practice and was not performed in the current study. As we observed a favorable outcome after intensifying immunosuppressive treatment, we hypothesize that these patients indeed had active disease at the time of scanning. It is important to note that PET scans do not differentiate active vasculitis from infections, as observed in 2 of our patients with PET-positive findings due to an underlying fungal infection. In one of these patients, a biopsy of a clinically occult lesion led to the discovery of cryptococcal myositis and masquerading vasculitis.¹⁸ The differentiation between infections and ANCA-mediated disease activity remains an area of uncertainty, especially because there is strong evidence that infections may be an important trigger in the multifactorial etiology of ANCA-associated vasculitis.¹⁹In the future, more sensitive diagnostic modalities, such as the combination of PET scanning with magnetic resonance imaging (PET/MRI), may identify the infectious foci, which started the cascade leading to the (re)activation of vasculitis.

Most importantly, PET scans revealed abnormalities during episodes of active disease in which ANCA were sometimes not detected and CRP levels not increased. However, more hotspots were observed if the CRP levels were elevated. In contrast, the highest observed SUVmax values were not related to CRP levels. These findings suggest that the disease may be more extensive, but not more severe, if biochemical parameters of inflammation are increased.

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Drug utilization, safety, and effectiveness of exenatide, sitagliptin, and vildagliptin for type 2 diabetes

Posted in Diabetes Mellitus, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Population Health Management, tagged DPP4 inhibitors and GLP1 receptor agonists, Incretin-mimetics and incretin-enhancers, Monitoring registry, Real clinical practice, type 2 diabetes on November 27, 2015| Leave a Comment »

Drug utilization, safety, and effectiveness of exenatide, sitagliptin, and vildagliptin for type 2 diabetes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Drug utilization, safety, and effectiveness of exenatide, sitagliptin, and vildagliptin for type 2 diabetes in the real world: Data from the Italian AIFA Anti-diabetics Monitoring Registry

S. Montilla, G. Marchesini, A. Sammarco, M.P. Trotta, P.D. Siviero, C. Tomino, D. Melchiorri, L. Pani for the AIFA Anti-Diabetes Monitoring Registry
Nutrition Diabetes and Cardiovasc Dis Dec 2014; 24(12):1346–1353 http://dx.doi.org/10.1016/j.numecd.2014.07.014

Background and aims

In Italy, the reimbursed use of incretin mimetics and incretin enhancers was subject to enrollment of patients into a web-based system recording the general demographic and clinical data of patients. We report the utilization data of glucagon-like peptide 1 (GLP1) receptor agonists and dipeptidylpeptidase-4 (DPP4) inhibitors in clinical practice as recorded by the Italian Medicines Agency (AIFA) Monitoring Registry.

Methods and results

From February 2008 to August 2010, 75,283 patients with type 2 diabetes were entered into the registry and treated with exenatide, sitagliptin, or vildagliptin. The treatment was administered to patients in a wide range of ages (≥75 years, n = 6125 cases), body mass index (BMI) (≥35 kg/m², n = 22,015), and metabolic control (HbA_1c ≥ 11% ((96 mmol/mol), n = 3151). Overall, 1116 suspected adverse drug reactions were registered, including 12 cases of acute pancreatitis (six on exenatide). Hypoglycemic episodes mainly occurred in combination with sulfonylureas. Treatment discontinuation for the three drugs (logistic regression analysis) was negatively associated with the male gender and positively with baseline HbA_1c, diabetes duration, and, limitedly to DPP-4 inhibitors, with BMI. Treatment discontinuation (including loss to follow-up, accounting for 21–26%) was frequent. Discontinuation for treatment failure occurred in 7.7% of cases (exenatide), 3.8% (sitagliptin), and 4.1% (vildagliptin), respectively, corresponding to 27–40% of all discontinuations, after excluding lost to follow-up. HbA_1c decreased on average by 0.9–1.0% (9 mmol/mol). Body weight decreased by 3.5% with exenatide and by 1.0–1.5% with DPP-4 inhibitors.

Conclusions

In the real world of Italian diabetes centers, prescriptions of incretins have been made in many cases outside the regulatory limits. Nevertheless, when appropriately utilized, incretins may grant results at least in line with pivotal trials.

Article Outline

A progressive intensification of treatment is mandatory in type 2 diabetes whenever lifestyle intervention fails to maintain metabolic control [1]. All major guidelines agree on administering metformin as the initial treatment, when tolerated and not contraindicated, but there is no consensus on second-line add-on treatment, in the case of unsatisfactory metabolic control. [[2], [3], [4], [5]].

In the past decade, injectable glucagon-like peptide-1 receptor agonists (GLP-1RAs) and orally administered inhibitors of dipeptidylpeptidase-4 (DPP-4Is) entered the diabetes arena [[6], [7]]. Since the initial marketing authorization as add-on therapies, these drugs have been granted extension of indications to include first-line monotherapy and combination with insulin. However, their best place in therapy remains uncertain [8]. In controlled clinical trials, both GLP-1RAs and DPP-4Is, combined with metformin, produce similar improvements in glycemic control as other second-line treatments, with no negative effects on body weight and overall hypoglycemia [[9], [10]]. However, only a few systematic analyses of long-term clinical data are available on large patients’ cohorts [[11], [12]], capturing treatment effects and prescription trends in the community.

In February 2008, the Italian Medicines Agency (AIFA) approved the reimbursed use of exenatide, sitagliptin, and vildagliptin, subject to enrollment of patients into a web-based system to monitor the appropriateness of use, safety profile, and effects on metabolic control and body weight. We report the results of the first 30-month monitoring, as derived from the AIFA Monitoring Registry. Of note, fixed-dose associations of sitagliptin and vildagliptin with metformin were made available along the years; in the present report, their use is considered equivalent to the combination use of the individual compounds. Focus is given to the clinical characteristics of patients, drug safety, and reasons for treatment discontinuation. An analysis of the percentage of patients reaching HbA_1c targets over time is also provided, to help clinicians tailor treatment on patients’ characteristics.

Patient population and baseline characteristics

A total of 77,864 records (38,811 on sitagliptin, 21,064 on exenatide, and 17,989 on vildagliptin), corresponding to 75,283 patients, were registered by 3741 diabetes specialists in 1278 centers, either hospital (n = 790) or community based (n = 488), distributed throughout Italy. On average, 16.5/10,000 inhabitants aged ≥18 were included (from 8.2 to 28.8 in different Italian regions).

The patients belonged to a fairly heterogeneous group, including a high proportion of cases scarcely represented in the trials supporting the marketing authorization of the three medicinal products. Over 50% of cases on exenatide and approximately 20% on DPP4-Is had severe obesity (BMI ≥ 35 kg/m²); exenatide patients exhibited higher median HbA_1c and a greater percentage of cases with very poor metabolic control (HbA_1c ≥ 11%, ≥97 mmol/mol). Elderly patients (≥75 years, n = 6125) constituted approximately 10% of the DPP-4I-treated cases (Table 1A; Supplemental Figure S2).

Table 1ABaseline demographic/clinical data of the population with diabetes enrolled in the AIFA Anti-diabetics Monitoring Registry with glucose-lowering agents.
Exenatide (n = 21,064)		Sitagliptin (n = 38,811)		Vildagliptin (n = 17,989)
Mean	SD	Mean	SD	Mean	SD
Age (years)	58.9	9.9	61.7	10.4	61.9	10.4
Duration of diabetes (years)	10.0	15.4	9.1	7.1	8.2	6.5
Body mass index (kg/m²)	36.1	6.8	30.8	5.7	30.5	5.5
Waist circumference (cm)	115.9	14.4	104.6	13.1	104.4	12.6
Fasting glucose (mg/dL)	187.8	49.8	170.8	41.6	171.9	41.1
HbA_1c (%) [mmol/mol]	8.8 [73]	1.3 [14]	8.3 [67]	1.1 [12]	8.2 [66]	1.1 [12]
Fasting C-peptide (ng/mL)	3.2	1.6	3.0	1.6	3.3	1.7
N	%	N	%	N	%
Male gender	10,109	48.0	20,446	52.7	9741	54.1
Age > 75 years	723	3.4	3666	9.4	1736	9.7
BMI > 35	10,835	51.4	7870	20.3	3300	18.3
HbA_1c > 11% (>97 mmol/mol)	1496	7.1	1139	2.9	516	2.9

Metformin was the background therapy in most cases, with/without concomitant sulfonylureas. Glitazones were rarely used, reflecting the Italian market. Monotherapy with sitagliptin was registered in <1% of cases (Table 1B).

Table 1BAssociation with other glucose-lowering agents.
Exenatide (n = 21,064)		Sitagliptin (n = 38,811)		Vildagliptin (n = 17,989)
N	%	N	%	N	%
No association^a	0	0	3.87	0.1	0
Metformin	10,691	50.8	25,116	64.7	15,289	85
Sulfonylureas	1323	6.3	1843	4.7	2062	11.5
Sulfonylureas + metformin	9050	43.0	9824	25.3	–^a	–^a
Glitazones	–^a	–^a	1624	4.2	638	3.5
Repaglinide	1450	6.9	276	0.7	–^a	–^a
Acarbose	260	1.2	225	0.5	72	0.4

In individual cases, background therapy could vary in the course of the observation. Please note that patients could be treated with more than one active principle; therefore, the sum of the percentages of cases may exceed 100%.

aOff-label according to marketing authorization.

Adverse drug reactions

During the 30-month observation period, 1116 ADRs were registered. The median time to ADR was 2.06, 2.85, and 3.87 months on exenatide, sitagliptin, and vildagliptin, respectively. Complete and partial recovery was observed in 717 and 179 cases, respectively; 103 cases did not recover, and late complications were registered in 13. No follow-up was available in 102 cases and two patients died. ADRs did not lead to treatment discontinuation only in 90 cases; after stopping the treatment, drug use was restarted in 100 cases.

ADRs were classified as severe in 77 cases (6.9%), particularly with exenatide (six acute pancreatitis, seven vomiting/nausea, and four renal failures, corresponding to an IR of 0.334, 0.390, and 0.223/1000 person-years, respectively) (Table 2). Three cases of acute pancreatitis occurred on sitagliptin and three more on vildagliptin (IRs: 0.097 and 0.221/1000 person-years, respectively). In addition, non-severe pancreatitis/elevated pancreatic enzymes were recorded in 48 cases (19 with exenatide, 16 with sitagliptin, and 13 with vildagliptin).

Table 2List of all severe ADRs and corresponding IR (in 1000 person-years) reported in the AIFA Anti-diabetics Monitoring Registry.
Event	Exenatide			Sitagliptin			Vildagliptin
Event	No.	IR^a	95% CI	No.	IR^a	95% CI	No.	IR^a	95% CI
Acute pancreatitis	6	0.334	(0.157–0.650)	3	0.097	(0.035–0.234)	3	0.221	(0.080–0.533)
Vomiting/nausea	7	0.390	(0.192–0.727)	1	0.032	(0.008–0.119)	0	(0.000–0.185)
Renal failure	4	0.223	(0.090–0.488)	0	(0.000–0.081)	1	0.074	(0.018–0.272)
Colon cancer	1	0.056	(0.013–0.205)	2	0.065	(0.020–0.180)	1	0.074	(0.018–0.272)
Epileptic convulsions	2	0.111	(0.034–0.310)	0	(0.000–0.081)	0	(0.000–0.185)
Abdominal pain	2	0.111	(0.034–0.310)	0	(0.000–0.081)	0	(0.000–0.185)
Severe hypoglycemia	1	0.056	(0.013–0.205)	1	0.032	(0.008–0.119)	0	(0.000–0.185)
Pneumonia	0	(0.000–0.140)	2	0.065	(0.020–0.180)	0	(0.000–0.185)
Breast cancer	1	0.056	(0.013–0.205)	2	0.065	(0.020–0.180)	0	(0.000–0.185)
Visual loss	0	(0.000–0.140)	1	0.032	(0.008–0.119)	0	(0.000–0.185)
Colon adenoma	0	(0.000–0.140)	0	(0.000–0.081)	1	0.074	(0.018–0.272)
Anaphylactic reaction/shock	1	0.056	(0.013–0.205)	1	0.032	(0.008–0.119)	0	(0.000–0.185)
Anemia	0	(0.000–0.140)	0	(0.000–0.081)	1	0.074	(0.018–0.272)
Cardiac failure	1	0.056	(0.013–0.205)	0	(0.000–0.081)	0	(0.000–0.185)
Atrioventricular block	1	0.056	(0.013–0.205)	0	(0.000–0.081)	0	(0.000–0.185)
Renal carcinoma	2	0.111	(0.034–0.310)	0	(0.000–0.081)	0	(0.000–0.185)
Cervix carcinoma	1	0.056	(0.013–0.205)	0	(0.001–0.081)	0	(0.000–0.185)
Coronary disease/Infarction	2	0.111	(0.034–0.310)	0	(0.000–0.081)	0	(0.000–0.185)
Cholecystitis	0	(0.000–0.140)	0	(0.000–0.081)	1	0.074	(0.018–0.272)
Cholestasis	0	(0.000–0.140)	1	0.032	(0.008–0.119)	0	(0.000–0.185)
Acute dermatitis	1	0.056	(0.013–0.205)	0	(0.000–0.081)	1	0.074	(0.018–0.272)
Gastric hemorrhage	0	(0.000–0.140)	1	0.032	(0.008–0.119)	0	(0.000–0.185)
Abdominal hernia	1	0.056	(0.013–0.205)	0	(0.000–0.081)	0	(0.000–0.185)
Atrial fibrillation	1	0.056	(0·013–0.205)	0	(0.000–0.081)	0	(0.000–0.185)
Liver dysfunction	0	(0.000–0.140)	0	(0.000–0.081)	2	0.147	(0.046–0.411)
Acute gastroenteritis	1	0.056	(0.013–0.205)	0	(0.000–0.081)	0	(0.000–0.185)
Congestive gastropathy	1	0.056	(0.013–0.205)	0	(0.000–0.081)	0	(0.000–0.185)
Ictus/cerebral hemorrhage/ischemia	1	0.056	(0.013–0.205)	1	0.032	(0.008–0.119)	1	0.074	(0.018–0.272)
Leukemia/lymphoma	0	(0.000–0.140)	2	0.065	(0.020–0.180)	1	0.074	(0.018–0.272)
Urticaria	2	0.111	(0.034–0.310)	0	(0.000–0.081)	0	(0.000–0.185)
Bladder cancer	0	(0.000–0.140)	0	(0.000–0.081)	1	0.074	(0.018–0.272)
Pericardial effusion	0	(0.000–0.140)	1	0.032	(0.008–0.119)	0	(0.000–0.185)
Gastric ulcer	1	0.056	(0.013–0.205)	0	(0.000–0.081)	0	(0.000–0.185)
Other	2	0.111	(0.034–0.310)	1	0.032	(0.008–0.119)	0	(0.000–0.185)
Total	43	2.397	(1.781–3.162)	20	0.645	(0.421–0.960)	14	1.034	(0.619–1.639)

aIncidence rate (IR) = # event (N)/person-time at risk (T).

Hypoglycemic episodes were reported in 1085 exenatide-treated patients, 608 on sitagliptin, and 207 on vildagliptin, with IRs of 20.6, 6.3, and 4.6/1000 person-years, respectively. Sulfonylureas, either alone or combined with metformin, increased the risk of hypoglycemia. The RR during add-on to sulfonylureas, compared with add-on to metformin, was 2.96 (95% confidence interval (CI), 2.33–3.50) on exenatide, 2.99 (95% CI, 2.45–3.64) on sitagliptin, and 1.84 (95% CI, 1.20–2.69) on vildagliptin. In add-on to sulfonylurea + metformin, the RRs further increased to 3.76 (95% CI, 3.24–4.36) and 2.94 (95% CI, 2.39–3.61) for exenatide and sitagliptin, respectively (not authorized for vildagliptin).

……………..

Effect on glycemic control and body weight

On exenatide, absolute HbA_1c decreased on average by 0.99% (0.9 mmol/mol) and body weight by 3.5% from baseline to the last available follow-up. The corresponding variations for sitagliptin and vildagliptin were −0.88% and −0.94% (0.8–0.9 mmol/mol) for HbA_1c, and around −1.0% for body weight. The probability of reaching the HbA_1c target of 7% (53 mmol/mol) or the secondary target of 8% (64 mmol/mol), after 3–4 or 8–9 months, decreased rapidly with increasing baseline HbA_1c, with <20% probability for baseline values >9% (>75 mmol/mol) (Fig. 1). The number of cases at target with baseline HbA_1c >11% was much lower for sitagliptin and vildagliptin than for exenatide, and the confidence interval of the estimate much larger.

Figure 1

Probability of achieving the targets of metabolic control (HbA_1c <7%, lower lines; <8%, upper lines) at 3–4 months (continuous lines) or 8–9 months (broken lines) as function of entry HbA_1c values.

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In the subset of centers compliant to follow-up, the probability of achieving the desired target was not dependent on age or BMI, but it was inversely related to baseline HbA1c and to the use of incretin mimetics/DPP-4Is as third-line therapy. The add-on to metformin and treatment duration (not on vildagliptin) increased the probability of reaching the target (Supplementary Table 2).

The AIFA Monitoring Registry of exenatide, sitagliptin, and vildagliptin, collecting data on the use, safety, and effectiveness of incretin mimetics/DPP-4Is, represents a significant step forward in the post-marketing evaluation of new or innovative medicines.

The safety profiles of exenatide, sitagliptin, and vildagliptin in Italian clinical practice were similar to those recorded in registration trials and recently reviewed [12]. Although favored by online registration, the total number of ADRs was relatively low – but much higher than that usually observed in post-marketing surveillance – despite the old age of the population, and no unexpected ADRs were registered, with only one case of heart failure with DPP-4Is [13]. The decision of the regulatory Italian Agency (AIFA) to limit the reimbursement of incretin-based therapies to diabetes specialists in a well-defined monitoring system might have favored an accurate selection of patients also in the community setting, limiting adverse reactions.

Two ADRs are of particular significance: pancreatitis and hypoglycemia. The association of exenatide and sitagliptin with pancreatitis was documented since 2006 and prompted close monitoring [[14], [15]]. Later, the potential risk appeared to be increased by diabetes per se; post-approval studies have documented cases associated with incretin use, but a causal relationship between treatment and pancreatitis was neither proved nor excluded [[16], [17], [18], [19], [20]]. In the registry, a few additional reports of non-severe pancreatitis or simply raised levels of pancreatic enzymes were also recorded, without differences between drugs. When these non-adjudicated ADRs were summed up to severe pancreatitis, the total incidence of pancreatic events was in the range reported in the general population with diabetes and should be considered in the context of the notoriety bias generated by alerts. A 2013 comprehensive review of preclinical and clinical data on pancreatic safety by the European Medicines Agency concluded that the concerns on the risk of pancreatitis should not be minimized [21]. Later, the publication of two large cardiovascular outcome DPP-Is trials [[13], [22]] and epidemiological data [23] stifled the debate; a 2014 joint Food and Drug Administration (FDA)–European Medicines Agency (EMA) assessment concluded with a low-risk [24] but suggested continuous capture of data.

As expected, exenatide and DPP4-I add-ons to metformin were accompanied by low rates of hypoglycemia [25]. On the contrary, a two-to threefold increase in hypoglycemia was observed in combination with sulfonylureas, both with and without metformin, but very few cases were recorded as severe ADRs, requiring hospital admission. These data are in keeping with registration studies and with recent clinical trials showing that DPP4-Is are associated with very low rates of hypoglycemia when combined with metformin [26], despite similar or only moderately inferior glucose-lowering efficacy compared to sulfonylureas.

The analysis of discontinuation rates and metabolic effects may give hints for an appropriate use of these drugs in the community. This approach seems sound, as confirmed by a sensitivity analysis in a subset of selected centers with adherence to follow-up ≥80% (Supplementary Tables 1 and 2). As expected, the discontinuation rates of all drugs increased systematically with higher baseline HbA_1c. They also increased with age for exenatide, not for gliptins, indicating a preferential use of oral agents in elderly subjects for whom a less strict metabolic target may be preferred [[3], [4], [27]]. On the contrary, weight loss might be the reason for the lower discontinuation rates of exenatide with increasing BMI, despite injections and higher baseline HbA_1c.

Two subpopulations, with limited safety data in registration studies, deserve particular attention. The AIFA Registry included many patients aged ≥70; in a few of them, gastrointestinal symptoms associated with exenatide were the precipitating factors of acute renal failure, a side effect to be considered in frail patients. DPP-4Is were demonstrated to be safe in a meta-analysis on patients aged ≥65, as well as in a systematic review, and vildagliptin was shown to be effective and safe also in subjects with diabetes aged ≥75 [[6], [9],[27]]. Future analyses of the elderly Italian cohort will throw light on the efficacy of DPP-4I in the elderly. Similarly, the very large group with morbid obesity in the AIFA Registry will offer a unique opportunity to test the effects of incretin-based therapies in these patients, where metabolic control remains difficult and the use of insulin may be critical, because it further increases body weight.

In our database, the effectiveness of incretin-based add-on therapies on HbA_1c and body weight was similar to that reported in a review of head-to-head trials [28], but these results should be taken with caution, considering that the high rate of L-FUs inflates effectiveness. HbA_1c was reduced on average by 0.9–1.0% (9 mmol/mol) in the general dataset, also in relation to HbA_1c at baseline, with much larger effects in subjects with poor metabolic control. In the AIFA Registry, exenatide and DPP-4Is were also prescribed to subjects with very poor metabolic control, above the levels where insulin is recommended by international guidelines [4]. Such prescribing approach may be explained by the opportunity to test these new drugs across the whole spectrum of disease, or as an extreme attempt before prescribing insulin. Fig. 1 provides an immediate picture of the possibility of attaining specific HbA_1c targets with incretin-based therapies in clinical practice, emphasizing the predictive value of baseline metabolic control. This figure may help clinicians forecast the results of treatment in their next patient, as modulated by other variables (i.e., age, BMI, diabetes duration, and background treatment), as reported in Supplementary Table 2. The observation that several patients with HbA_1c in the range 9–11% (75–97 mmol/mol) may reach an acceptable metabolic control with a low incidence of adverse reactions, including hypoglycemic events, is clinically relevant. Drug effectiveness should always be considered in the context of existing therapies [29], safety, cost, therapeutic inertia [30], and the beneficial effects of intensive lifestyle counseling, which remains mandatory at any step of intensified treatment. Notably, in frail patients, a patient-centered approach and progressively less challenging targets are proposed by international guidelines, to avoid the risk of adverse events. [4].

Our study presents limitations and strengths. First, the major limitation is an observation period of only 30 months, too short to draw definite conclusions on long-term efficacy (i.e., effects on diabetic complications). Second, due to its observational nature, baseline differences, and high rates of L-FU, any comparisons of safety, discontinuation, and effect on metabolic and weight control among the three drugs should be made with extreme caution. Third, given the purpose of the AIFA Registry, there was no comparator-treated group. Conversely, the main strength is the very large and heterogeneous diabetes cohort, including the complete dataset from an entire European nation, where drugs were used under strict regulatory access, requiring online registration for reimbursement.

In conclusion, data on the compliance, safety, and effectiveness of incretin-based therapies derived from the AIFA Registry, while not capturing any new safety signal, provide a comprehensive framework for health-care providers to regulate the use of these drugs in the community. These data might be useful to address several important points, including the independent effect of baseline HbA_1c on its decline, the safety and effectiveness in subjects with diabetes over 75, and the effectiveness of incretins – also including liraglutide and saxagliptin from August 2010 – in the large cohort of obese subjects with BMI >35. These analyses will be carried out when the monitoring data will be available in the new and updated in-house web platform currently being developed. Whenever effective strategies of lifestyle changes preliminary to any further step in treatment intensification fail, the implementation of new treatments, including incretin-based therapies, should be dictated by solid data on long-term safety and effectiveness in the context of available drugs for type 2 diabetes, favoring a patient-centered approach. [4].

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HDL oxidation in type 2 diabetic patients

Posted in Acute Myocardial Infarction, Chemical Biology and its relations to Metabolic Disease, Curation, Diabetes Mellitus, Disease Biology, tagged Apolipoprotein A-I, HDL oxidation, type 2 diabetes on November 27, 2015| Leave a Comment »

HDL oxidation in type 2 diabetic patients

Larry H. Bernstein, MD, FCAP, Curator

LPBI

High-density lipoprotein oxidation in type 2 diabetic patients and young patients with premature myocardial infarction

G. Sartore ^a, R. Seraglia ^b, S. Burlina ^a^,^,, A. Bolis ^a, R. Marin ^a, E. Manzato ^a, E. Ragazzi ^c, P. Traldi ^b, A. Lapolla^{a

Nutr Metab Cardiovasc Dis April 2015; 25(4): 418–425 http://dx.doi.org:/10.1016/j.numecd.2014.12.004}

Highlights

• Oxidative damage can generate dysfunctional HDL reducing its anti-atherogenic role.
• Increased MetO levels in ApoA-I in patients with premature MI and in type 2 DM.
• An increase in MetO levels in ApoA-I could result in HDL dysfunction.

Background and aims

ApoA-I can undergo oxidative changes that reduce anti-atherogenic role of HDL. The aim of this study was to seek any significant differences in methionine sulfoxide (MetO) content in the ApoA-I of HDL isolated from young patients with coronary heart disease (CHD), type 2 diabetics and healthy subjects.

Methods and results

We evaluated the lipid profile of 21 type 2 diabetic patients, 23 young patients with premature MI and 21 healthy volunteers; we determined in all patients the MetO content of ApoA-I in by MALDI/TOF/TOF technique. The typical MALDI spectra of the tryptic digest obtained from HDL plasma fractions all patients showed a relative abundance of peptides containing Met¹¹²O in ApoA-I in type 2 diabetic and CHD patients. This relative abundance is given as percentages of oxidized ApoA-I (OxApoA-I). OxApoA-I showed no significant correlations with lipoproteins in all patients studied, while a strong correlation emerged between the duration of diabetic disease and OxApoA-I levels in type 2 diabetic patients.

Conclusions

The most remarkable finding of our study lies in the evidence it produced of an increased HDL oxidation in patients highly susceptible to CHD. Levels of MetO residues in plasma ApoA-I, measured using an accurate, specific method, should be investigated and considered in prospective future studies to assess their role in CHD.

No more than 25% of the risk of coronary heart disease (CHD) can be explained by known risk factors, despite their high prevalence [1].

High-density lipoprotein (HDL) protects artery wall from atherosclerosis, in particular they remove excess cholesterol from artery wall macrophages and carries it back to the liver for excretion in bile [2]. Apolipoprotein A-I (ApoA-I) is the main protein of HDL and it plays a crucial part in the first cholesterol transport reversal step by enhancing sterol efflux from macrophages [3].

Epidemiological studies have demonstrated that plasma HDL independently predict the risk of developing atherosclerosis and cardiovascular disease [4]. More recently, however, it has emerged that HDL quality also seems to be an important parameter in atheroprotection, though there is little data in the literature to support it [5].

An increasing body of evidence shows that HDL isolated from atheromas and the plasma of patients with established CHD lacks these anti-atherogenic properties [6]. HDL can be functionally deficient in populations at high risk of CHD, as in type 2 diabetes mellitus, due to glycation and oxidative changes in their HDL, apolipoproteins, and/or enzymes[7].

ApoA-I in particular can undergo oxidative changes that reduce its anti-atherogenic role[8]. Oxidation of the Tyr and Met residues in ApoA-I by myeloperoxidase drastically impairs the protein’s ability to promote cholesterol efflux via the ABCA1 pathway [9]. Levine and colleagues [10] suggested that Met residues in protein serve as endogenous antioxidants, protecting functionally important amino acids against oxidation. In ApoA-I in particular, Met⁸⁶ and Met¹¹² are thought to be important for cholesterol efflux, and Met¹⁴⁸is believed to be involved in LCAT activation [11].

Brock et al. recently examined the extent and sites of methionine sulfoxide (MetO) formation in the ApoA-I of HDL isolated from the plasma of healthy controls and type 1 diabetic subjects, demonstrating that MetO formation was significantly greater in diabetic patients than in a control group at all three sites considered (Met⁸⁶, Met¹¹², and Met¹⁴⁸)[12].

Considering the relevant role of HDL oxidation in the onset of atherosclerotic processes, we ran a pilot study on a small group of type 2 diabetic patients and young people prematurely experiencing acute myocardial infarction (MI): in both these groups we found higher levels of Met¹¹²O than in healthy controls [13]. That investigation was carried out by microfluidic-LC/ESI-MS measurements. In a further study the determination of MetO content of ApoA-I in type 2 diabetic patients was performed by MALDI/MS [14] and the results obtained perfectly overlap those achieved in the previous LC/MS investigation. These results proved that possible oxidation phenomena, sometimes observed in MALDI conditions [15], are in this case absent.

The aim of this study was to assess larger study groups to seek any significant differences in MetO between patients with premature MI, type 2 diabetics and healthy subjects, and to identify any correlations with these individuals’ lipoproteins. A secondary aim was to see whether the duration of the diabetic patients’ disease correlated with HDL oxidation.

MALDI/MS

MALDI/time of flight (TOF) and MALDI/TOF/TOF measurements were performed using a MALDI/TOF/TOF UltrafleXtreme instrument (Bruker Daltonics, Bremen, Germany), equipped with a 1 kHz smartbeam II laser (λ = 355 nm) and operating in the positive reflectron ion mode. The instrumental conditions were: IS1 = 25 kV; IS2 = 21.65 kV; reflectron potential = 26.3 kV; delay time = 0 nsec. The matrix was α-cyano-4-hydroxycinnamic acid (HCCA) (saturated solution in H₂O/acetonitrile (50:50; v/v) containing 0.1% TFA). Five μL of purified tryptic digest and 5 μL of matrix solution were mixed together, then 1 μL of the resulting mixture was deposited on the stainless steel sample holder and allowed to dry before placing it in the mass spectrometer. External mass calibration (Peptide Calibration Standard) was based on monoisotopic values of [M+H]+ of angiotensin II, angiotensin I, substance P, bombesin, ACTH clip [1], [2], [3], [4],[5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16] and [17], ACTH clip (18–39), somatostatin 28 at m/z 1046.5420, 1296.6853, 1347.7361, 1619.8230, 2093.0868, 2465.1990 and 3147.4714. TOF/TOF experiments were performed using the LIFT device in the following experimental conditions: IS1: 7.5 kV; IS2: 6.75 kV; Lift1: 19 kV; Lift2: 3.7 kV; Reflector1: 29.5 kV; delay time: 70 ns.

Table 1 shows the demographic and clinical characteristics of patients and controls. The three groups were matched for age and smoke; the controls and diabetics were also matched for gender, while the premature MI group consisted almost entirely of men, with only one female patient. Type 2 diabetic patients were not in a situation of good metabolic control, their HbA1c levels being a mean 8.22 ± 0.84% and their FPG 156.7 ± 29.7 mg/dl.

Table 1.Clinical characteristics of type 2 diabetic patients, young patients with premature CHD and controls. Data are expressed as mean ± standard deviation. To assess statistical differences between groups, ANOVA followed by Tukey’spost-hoc test was used. ●●● p < 0.001; ●● p < 0.01; ● p < 0.05; ns: not significant;–:not applied. Abbreviations: FPG = fasting plasma glucose; TC = total cholesterol; LDL = low-density lipoprotein; HDL = high-density lipoprotein; OxApoAI = oxidized Apolipoprotein AI; MI = myocardial infarction.

Control subjects (C) (n = 21)	CHD patients (CHD) (n = 23)	Type 2 diabetic patients (D) (n = 21)	P
Control subjects (C) (n = 21)	CHD patients (CHD) (n = 23)	Type 2 diabetic patients (D) (n = 21)	C vsCHD	CvsD	CHDvs D
Gender (M/F)	6/15	22/1	10/11	–	–	–
Age (yrs)	41.4 ± 2.8	40.7 ± 3.4	51.8 ± 3.5	ns	ns	ns
Diabetes duration (yrs)	–	–	8.5 ± 3.9	–	–	–
FPG (mg/dl)	83.5 ± 4.8	89.1 ± 7.4	156.7 ± 29.7	ns	●●●	●●●
HbA1c (%)	5.2 ± 0.2	5.3 ± 0.2	8.2 ± 0.8	ns	●●●	●●●
TC (mg/dl)	203.2 ± 33.3	205.3 ± 26.3	203.2 ± 37.0	ns	ns	ns
LDL (mg/dl)	117.5 ± 30.6	143.7 ± 24.7	118.3 ± 30.6	●●	ns	●
HDL (mg/dl)	68.7 ± 11.2	38.3 ± 10.4	49.7 ± 14.0	●●●	●●●	●●
Triglycerides (mg/dl)	85.4 ± 21.6	146.3 ± 55.9	178.0 ± 91.8	●●	●●●	ns
ApoA1 (mg/dl)	148.2 ± 31.3	117.2 ± 14.5	128.9 ± 14.7	●	●	ns
OX ApoA1 (%)	1.7 ± 1.3	4.8 ± 2.6	10.6 ± 5.3	●	●●●	●●●
MI (no/yes)	21/0	0/23	21/0	–	–	–
Statin therapy (yes/no)	0/21	23/0	18/3	–	–	ns
Anti-platelet agents (yes/no)	0/21	23/0	17/4	–	–	ns
Antihypertensive drugs (yes/no)	0/21	23/0	20/1	–	–	ns
Smokers (yes/no/ex)	4/15/2	5/16/2	6/14/1	ns	ns	ns

Table options

The three groups had similar total cholesterol levels. The group of patients with a premature MI had the highest levels of LDL cholesterol and the lowest levels of HDL cholesterol. Their triglycerides were also higher than in the healthy controls, but lower than in the diabetic patients.

Characterization of Met¹¹² and Met¹¹²-O containing peptides

The typical MALDI spectra of the tryptic digest obtained from HDL plasma fractions of healthy subjects, diabetics and CHD patients are given in Fig. 1. MS/MS experiments performed on the two ions at m/z 1283.6 and 2645.4 showed that the sequences of the corresponding peptides are W¹⁰⁸QEEM¹¹²ELYR and V⁹⁷QPYLDDFQKKWQEEM¹¹²ELYR, both of which contain the methionine residue in position 112 (Met¹¹²). Looking at selected regions of the spectra related to the two above-mentioned ions, some differences appear between the healthy controls vs the diabetic patients and CHD patients. In the case of the diabetics and CHD patients, the two peaks at m/z 1299 and 2661 become more evident than those detected in the case of healthy subjects. These two peptides, differing from the above-described species by 16 Da, can be justified by the presence of the previously-mentioned peptides containing a Met¹¹²O moiety (see Fig. 2). MS/MS experiments performed on these two ions confirms this hypothesis, based on the presence of a fragment ion due to the loss of CH₃SOH. This result confirms that oxidation occurs at Met¹¹² in both the peptides. The above-described relative abundance of peptides containing Met¹¹²O- and Met¹¹² was ascertained for all samples. The percentages of OxApoA-I were calculated dividing the sum of the abundances of the peaks at m/z 1299 and 2661 (originating from oxidation of Met¹¹²) to the sum of the abundances of the four peaks of interest: the results so obtained are shown in Table 1. Both the diabetic and the CHD patients showed significantly higher OxApoA-I levels than controls. We did not observe any significant correlation between the levels of ApoA-I and OxApoA-I in all groups (controls: r = −0.031; diabetics: r = 0.092; CHD patients: r = 0.20, respectively).

: Figure 1.

The typical MALDI spectra of the tryptic digest obtained from HDL plasma fractions of healthy subjects, diabetic and CHD patients.; Figure options

: Figure 2.

Expanded view (A: m/z 1283–1299; B: m/z 2635–2690) of the MALDI mass spectra of tryptic digests from healthy subjects, diabetic patients and CHD patients.; Figure options

It is to underline that the possible ex-vivo oxidation of methionine residue was checked analyzing the lyophilized HDL samples after two and four months of storage at −30 °C. No significant variation in the content of Met¹¹²O was observed, indicating that ex-vivo oxidation is inhibited at storage temperature.

Correlations

OxApoA-I showed no significant correlations with lipoproteins, while there were inverse significant correlations between HDL cholesterol and triglycerides in both the diabetic and the CHD patients (p < 0.02), but not in the healthy controls, as shown in Table 2. No correlation emerged between the OxApoA-I and HbA1c levels in the diabetic patients (r = 0.0344).

Table 2.Linear correlation between oxidized ApoA-I (Ox-ApoA-I) and serum cholesterol in the three groups of patients. Data are the Pearson product–moment correlation coefficient (Pearson’s r) with the lower and upper 95% confidence intervals (in parentheses).*p < 0.02.

Correlation between variables	Control subjects, n = 21 (Lower and upper 95% C.I.)	CHD patients, n = 23 (Lower and upper 95% C.I.)	Diabetic patients, n = 21 (Lower and upper 95% C.I.)
Ox-ApoA-1 vs total cholesterol	0.3757 (−0.0669 ÷ 0.6947)	0.0519 (−0.3681 ÷ 0.4544)	−0.3287 (−0.6659 ÷ 0.1200)
Ox-ApoA-1 vs LDL-cholesterol	0.3557 (−0.0897 ÷ 0.6826)	−0.1688 (−0.5432 ÷ 0.2616)	−0.3193 (−0.6600 ÷ 0.1303)
Ox-ApoA-1 vs HDL-cholesterol	0.0745 (−0.3690 ÷ 0.4904)	0.3130 (−0.1139 ÷ 0.6423)	0.0839 (−0.3608 ÷ 0.4976)
Ox-ApoA-1vs triglycerides	0.1965 (−0.2570 ÷ 0.5790)	0.0434 (−0.3755 ÷ 0.4476)	−0.1953 (−0.5782÷0.2581)
Triglycerides vs HDL-cholesterol	−0.3973 (−0.7076 ÷ 0.0415)	−0.4898* (−0.7505 ÷ −0.0972)	−0.5413* (−0.7887 ÷ −0.1431)

Table options

In order to evaluate with a more integrated approach the presence of interrelationships among variables, the non-parametric technique of PCA was considered. The analysis was extended to the three groups as a whole, in order to check any distribution among the individuals, and the respective role of the considered variables. As the biplot of Fig. 3shows, it was confirmed the previously found lack of any relationship between the OxApoA-I levels and HDL cholesterol or triglycerides, and it was confirmed also the presence of an inverse correlation between HDL cholesterol or triglycerides; moreover, from this analysis a strong direct correlation between the duration of diabetic disease and OxApoA-I levels emerged.

: Figure 3.

Biplot of the first two principal components (PC1 and PC2) obtained by PCA conducted on the most representative variables from diabetic patients, CHD patients and controls.

In the present, small cross-sectional study, our data analyses support the impression that the atheroprotective effect of HDL may be deficient in patients experiencing a premature MI and in cases of type 2 DM, both models of accelerated atherosclerosis [23]. This HDL dysfunction could be due to an increase in MetO levels in ApoA-I. We demonstrated, not only that type 2 diabetic patients and young patients with premature acute MI share the same ApoA-I oxidation, but also and more importantly they both have a greater HDL oxidation than controls, irrespective of their HDL levels. This feature was recently observed in type 1 diabetic patients compared with healthy controls, and it may contribute to an accelerated atherosclerosis [12]. These findings provide a new clinical perspective compared to preliminary results obtained by microfluidic-LC/ESI-MS [13], this time using an alternative technique (MALDI/MS), that makes the analysis far less time-consuming, as we previously showed in type 2 diabetic patients and healthy controls [14]. Our group of type 2 diabetic patients showed no signs of CHD despite their more severely oxidized HDL. We surmise that they offset the higher levels of oxidized HDL with higher levels of HDL, so the ratio of HDL to oxidized HDL might be a better marker of CHD than low HDL levels. Unfortunately, since our method only allowed for a semiquantitative assessment of the oxidation of the above-described peptides, these data cannot be used to calculate the HDL/oxidized-HDL ratio.

It is worth noting that no correlation emerged between MetO levels in ApoA-I and HbA1c, indicating that ApoA-I oxidation appears unrelated to the degree of glycemic control. This finding is in agreement with previous observations that have shown no correlation between glyco-oxidation products, such as glyoxal and methylglyoxal, which better represent the real glyco-oxidative stress experienced by patients [24].

On the other hand, our data suggest that duration of disease might be the parameter most closely related to MetO levels in ApoA-I in type 2 diabetes. In this contest, the antioxidant system could play an important part in the onset of cardiovascular complications by counter-regulating the increased oxidative stress, as we found in various phenotypes of type 2 diabetic patients with and without micro- and macrovascular complications [25] and [26]. Several studies have also demonstrated that decreased levels of antioxidants favor cardiovascular disease in subjects without diabetes [27]. As regards our data on HDL oxidation, we hypothesized that the increase of Apo-AI oxidation could be due to the decreased levels of antioxidant defenses that characterized type 2 diabetic patients with long duration of disease and patient with premature MI. Recent observations, in fact, showed that serum myeloperoxidase/paraoxonase 1 ratio is a potential indicator of dysfunctional HDL and risk stratification in CHD [28]. At the end HDL oxidation process could be partially independent from oxidative stress burden, but affected by decreased antioxidant capacity.

As for the higher triglyceride levels found in our type 2 DM and CHD patients, we surmise that hypertriglyceridemia could be a prognostic marker even in young patients with premature MI, irrespective of other cardiovascular risk factors, as previously reported[29]. Both our groups of patients showed a strong inverse relationship between their HDL and triglyceride levels, a situation typical of insulin resistance and found associated with MI occurring before 40 years of age [30].

As regards LDL cholesterol, we found the highest level in young CHD patients who were all in statin therapy. Considering the very short period of statin therapy and knowing that to reach the full effect it needs at still a month, CHD patients showed LDL cholesterol levels not still at target. On the other side, statin therapy hardly have had an impact on the oxidation of HDL. In any case statin protective effect on the oxidation strengthen our conclusions.

All these quantitative and qualitative lipoprotein features (higher oxidized HDL, higher triglycerides and lower HDL levels) suggest the feasibility of characterizing patients at high risk of CHD in terms of their lipid profile, as illustrated in the integrated biplot ofFig. 3.

In conclusion, the most remarkable finding of our study lies in the evidence it produced of an increased HDL oxidation in patients highly susceptible to CHD. Levels of MetO residues in plasma ApoA-I, measured using an accurate, specific method, should be investigated and considered in prospective future studies in order to assess their possible role as a novel risk factor – and eventually as a therapeutic target – to reduce the burden of cardiovascular complications.

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Dipeptydil peptidase-4 inhibitors in type 2 diabetes

Posted in Clinical & Translational, Curation, Diabetes Mellitus, Metabolism, Pharmaceutical Analytics, tagged Dipeptidyl Peptidase-4 inhibitors, meta-analysis, Type 2 diabetes mellitus on November 27, 2015| Leave a Comment »

Dipeptydil peptidase-4 inhibitors in type 2 diabetes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Dipeptydil peptidase-4 inhibitors in type 2 diabetes: A meta-analysis of randomized clinical trials

M. Monami, I. Iacomelli, N. Marchionni, E. Mannucci

Unit of Geriatric Medicine, Department of Critical Care Medicine, University of Florence and Azienda Ospedaliera Careggi, Florence, Italy
Nutrition, Metabolism & Cardiovascular Diseases (NMCD) May 2010; 20(4):224–235 http://dx.doi.org/10.1016/j.numecd.2009.03.015

Background and Aim

The role of Dipeptidyl Peptidase-4 (DPP-4) inhibitors in the treatment of type 2 diabetes is debated; many recent trials, which were not included in previous meta-analyses, could add relevant information.

Methods and Results

All available randomized controlled trials (RCTs), either published or unpublished, performed in type 2 diabetic patients with DPP-4 inhibitors, with a duration >12 weeks were meta-analyzed for HbA1c, BMI, hypoglycemia, and other adverse events. A total of 41 RCTs (9 of which are unpublished) was retrieved and included in the analysis. Gliptins determine a significant improvement of HbA1c in comparison with a placebo (−0.7 [−0.8:−0.6]), with a low risk of hypoglycemia. DPP-4 inhibitors show a similar efficacy in monotherapy and in combination with other agents. The risk of cardiovascular events and all-cause death with DPP-4 inhibitors is 0.76 [0.46–1.28] and 0.78 [0.40–1.51], respectively.

Conclusions

DPP-4 inhibitors reduce HbA1c, although to a lesser extent than sulphonylureas, with no weight gain and no hypoglycemic risk; further data are needed to assess their long-term safety.

Oral Dipeptidyl Peptidase-4 (DPP-4) inhibitors sitagliptin [1] and vildagliptin [2], which increase circulating levels of Glucagon-Like Peptide-1 (GLP-1), have recently been approved for use in type 2 diabetes; other molecules of the same class (such as saxagliptin and alogliptin) are under development.

The role of those new drugs in the treatment of type 2 diabetes is debated. The consensus algorithm of the American Diabetes Association and the European Association for the Study of Diabetes [[3], [4]], in its revised version [4], suggests limiting the use of GLP-1 receptor agonists and DPP-4 inhibitors only in some specific cases, without considering those agents in the mainstream (“Tier 1”) of the algorithm. Conversely, DPP-4 inhibitors are not even included as a second choice, although their use is contemplated in selected patients. The reasons for this exclusion are their perceived limited efficacy on HbA1c in comparison with other agents, their poorly defined safety profile, and their cost [[3], [4]].

Efficacy and safety need to be assessed through a comprehensive review of currently available clinical trials. Some detailed reviews of published studies have been recently published [[1], [2], [5]]; furthermore, some meta-analyses have been performed [[1], [6], [7], [8]]. However, currently available meta-analyses included only published studies, without any attempt at retrieving data from completed and publicly disclosed, although not formally published, clinical trials. Furthermore, several trials have been published in the last few months, increasing in a relevant manner the available data base for the assessment of the clinical profile of DPP-4 inhibitors.

The aim of the present study is to offer a comprehensive and updated synthesis of all available clinical data on the safety and efficacy of DPP-4 inhibitors.

The trial flow is summarized in Fig. 1, and the characteristics of the trials included in the meta-analysis are summarized in Table 1. Among the trials included, 32 were described in publications in peer-reviewed journals; results of 9 unpublished trials were disclosed on different websites. Furthermore, 10 unpublished trials, the results of which were undisclosed, could be identified (Table 2). Notably, results could be retrieved for the large majority of trials on currently available DPP-4 inhibitors (sitagliptin and vildagliptin), while only results of preliminary phase II studies were available for products currently under development (saxagliptin).

Figure 1

Trial flow diagram. RCT: randomized clinical trial.

View Large Image | Download PowerPoint Slide

Table 1Characteristics of the studies included in the meta-analysis.
Study (Ref.)	Dose (mg/die)	Comparator	Add-on to	Description of randomization	Description of blinding	Reporting of drop-out	Intention-to-treat
Vildagliptin
Pan [33]	100	Acarbose	None	NA	NA	A	Yes
Schweizer [28]	100	Metformin	None	NA	NA	A	Yes
Rosenstock [34]	50–100	Rosiglitazone	None	NA	NA	A	Yes
2329 [14]	50–100	Pioglitazone	None	NR	NR	NR	Yes
Bolli [21]	100	Pioglitazone	Metformin	NA	NA	A	No
Rosenstock [35]	100	Pioglitazone	None	NA	A	A	Yes
Rosenstock [35]	50–100	Placebo	Pioglitazone	NA	A	A	Yes
Dejager [36]	50–100	Placebo	None	NA	NA	A	Yes
Scherbaum [37]	50	Placebo	None	NA	NA	A	Yes
Mari [38]	50	Placebo	None	NA	NA	A	NR
Scherbaum [39]	50	Placebo	None	NA	NA	A	Yes
Pratley [27]	50	Placebo	None	NA	A	A	Yes
Pi-Sunyer [40]	50–100	Placebo	None	NA	NA	A	Yes
Ristic [41]	25–100	Placebo	None	NA	NA	NA	Yes
1202 [14]	20–100	Placebo	None	NR	NR	NR	Yes
Ahren [42]	50	Placebo	Metformin	NA	NA	A	NR
Bosi [22]	50–100	Placebo	Metformin	NA	NA	A	Yes
Garber [43]	50–100	Placebo	Pioglitazone	NA	NA	A	Yes
Garber [19]	50–100	Placebo	Glimepiride	A	NA	A	Yes
1302 [14]	100	Placebo	Glimepiride	NR	NR	NR	Yes
Fonseca [20]	100	Placebo	Insulin	NA	NA	A	Yes
1303 [14]	50–100	Placebo	NR	NR	NR	NR	Yes
D’Alessio [44]	100	Placebo	Metf./None	NA	NA	A	Yes

Sitagliptin
PN-036 [15]	50–100	Metformin	None	NA	A	A	Yes
Scott [45]	100	Rosiglitazone	Metformin	NA	NA	A	Yes
Scott [45]	100	Placebo	Metformin	NA	NA	A	Yes
PN-035 [15]	100	Pioglitazone	Glim±Met	NA	NA	A	Yes
Nauck [17]	100	Glipizide	Metformin	NA	NA	A	Yes
PN-028 [15]	25–50	Placebo/Glip.	OAD/Insulin	NR	NR	NR	Yes
Scott [18]	10–100	Glipizide	None	A	A	A	Yes
Scott [18]	10–100	Placebo	None	A	A	A	Yes
Nonaka [46]	100	Placebo	None	NA	NA	A	Yes
Hanefeld [16]	25–100	Placebo	None	NA	A	A	No
Raz [47]	100–200	Placebo	None	NA	NA	A	Yes
Goldstein [23]	50–100	Placebo	None	NA	A	A	Yes
Rosenstock [35]	100	Placebo	Pioglitazone	NA	NA	A	Yes
Hermansen [24]	100	Placebo	Glim±Metf	NA	NA	A	Yes
Goldstein [23]	50–100	Placebo	Metformin	NA	A	A	Yes
Charbonnel [48]	100	Placebo	Metformin	NA	NA	A	Yes
Aschner [49]	100–200	Placebo	None	NA	NA	A	Yes
Raz [50]	100	Placebo	Metformin	A	NA	A	Yes
PN-040 [15]	100	Placebo	OAD/None	NR	NR	NR	Yes
PN-044 [15]	25–200	Placebo	OAD/None	NR	NR	NR	Yes

Saxagliptin
Rosenstock [51]	2.5–40	Placebo	None	NA	NA	A	Yes

NA: not adequate or not adequately reported; A: adequate; NR: not reported; glip.: glipizide; glim±metf: glimepiride and/or metformin; metf.: metformin; OAD: oral antidiabetic drugs; and SU/metf: sulfonylureas or metformin.

Table 2Characteristics of the unpublished and undisclosed studies.
Study	# Patients planned	Comparator	Add-on to	Trial duration (weeks)	Design	Randomization	Study end date^a
DPP-4 inhibitors

Vildagliptin
NCT00368134 [52]	370	Voglibose	None	12	PS	Double blind	June 2007
NCT00396227 [52]	2665	Glitazones	Metformin	12	PS	Open label	October 2007

Sitagliptin
NCT00411554 [52]	310	Voglibose	None	12	PS	Double blind	August 2007

Saxagliptin
NCT00327015 [52]	1396	Placebo	Metformin	52	PS	Double blind	November 2007
NCT00327015 [52]		Metformin	None	52	PS	Double blind
NCT00121641 [52]	460	Placebo	None	24	PS	Double blind	August 2007
NCT00374907 [52]	36	Placebo	None	12	PS	Double blind	October 2007
NCT00295633 [52]	555	Placebo	Glitazones	24	PS	Double blind	October 2007
NCT00121667 [52]	720	Placebo	Metformin	24	PS	Double blind	August 2006
NCT00313313 [52]	780	Placebo	Glyburide	24	PS	Double blind	September 2007
NCT00316082 [52]	365	Placebo	None	24	PS	Double blind	November 2007

PS: parallel series.

aFinal data collection date for primary outcome measure.

The Begg adjusted rank correlation test (Kendall tau: −74; p=0.13) and the Egger regression approach (intercept, −2.81 [CI, –6.91–1.27]) suggested no major publication bias.

…………….

Table 3Moderators and outcome variables in individual studies included in the meta-analysis.
Study (Ref.)	# Patients (ID/C)	Comparator	Trial duration (weeks)	>Age^a(years)	Duration of DM^a(years)	HbA1c baseline^a(%)	HbA1c endpoint (%, ID/C)	BMI baseline^a(Kg/m²)	BMI endpoint (Kg/m²)
DPP-4 inhibitors

Vildagliptin
Pan [33]	440/220	Acarbose	24	52	1.2	8.6	7.2/7.3	26.1	26.3/25.2
Schweizer [28]	526/254	Metformin	52	53	1.0	8.7	7.7/7.3	32.4	32.5/31.8
Rosenstock [34]	459/238	Rosiglitazone	24	54	2.5	8.7	7.6/7.4	32.5	32.1/33.5
2329 [14]	218/55	Pioglitazone	12	52	2.0	10.0	NR	NR	NR
Bolli [21]	295/280	Pioglitazone	24	56	6.4	8.4	7.5/7.5	32.1	32.1/32.8
Rosenstock [35]	154/161	Pioglitazone	24	51	2.0	8.7	7.0/7.3	29.4	29.9/29.4
Rosenstock [35]	292/161	Placebo	24	52	2.0	8.7	7.5/7.3	29.3	29.5/29.4
Dejager [36]	472/160	Placebo	24	54	2.1	8.4	7.6/8.1	32.9	NR
Scherbaum [37]	67/61	Placebo	52	64	3.3	6.6	6.6/7.1	30.2	NR
Mari [38]	156/150	Placebo	52	63	2.6	6.7	6.5/6.9	30.2	NR
Scherbaum [39]	156/150	Placebo	52	63	2.5	6.7	6.5/6.9	30.2	30.2/29.9
Pratley [27]	70/28	Placebo	12	55	4.0	8.0	7.4/8.1	29.9	NR
Pi-Sunyer [40]	262/92	Placebo	24	51	2.1	8.4	7.7/8.4	32.2	31.9/32.2
Ristic [41]	221/58	Placebo	12	56	3.0	7.7	7.2/7.7	31.1	31.0/31.4
1202 [14]	219/72	Placebo	12	59	NR	7.4	6.7/7.	24.0	NR
Ahren [42]	56/51	Placebo	12	57	5.5	7.8	7.1/7.8	29.7	NR
Bosi [22]	349/171	Placebo	24	54	6.2	8.4	7.5/8.4	32.7	32.5/31.7
Garber [43]	260/138	Placebo	24	54	4.7	8.7	7.6/8.1	32.4	NR
Garber [19]	264/144	Placebo	16	58	7.1	8.5	7.9/8.6	31.4	31.8/31.2
1302	102/100	Placebo	12	60	9.0	7.9	6.8/7.9	NR	NR
Fonseca [20]	144/152	Placebo	24	59	14.7	8.4	7.9/8.2	33.1	33.8/33.1
1303 [14]	178/61	Placebo	12	60	6.5	7.4	6.5/7.7	NR	NR
D’Alessio [44]	20/19	Placebo	12	55	3.5	6.7	6.3/6.3	32.3	NR

Sitagliptin
PN-036 [15]	179/176	Metformin	30	53	4.5	8.9	8.1/7.6	31.9	NR
Scott [45]	94/87	Rosiglitazone	18	55	5.0	7.7	7.0/6.9	30.2	30.1/30.9
Scott [45]	94/92	Placebo	18	55	5.0	7.7	7.0/7.5	30.1	30.1/29.8
PN-035 [15]	91/68	Pioglitazone	30	56	8.7	8.2	7.6/8.0	31.2	NR
Nauck [17]	576/559	Glipizide	52	57	6.3	7.7	7.2/7.0	31.2	30.7/31.7
PN-028 [15]	65/26	Placebo/Glip	54	68	13.5	7.7	7.0/7.6	NR	NR
Scott [18]	595/123	Glipizide	12	55	5.0	7.9	7.5/7.1	30.8	NR
Scott [18]	595/125	Placebo	12	55	5.0	7.9	7.5/8.1	31.0	NR
Nonaka [46]	75/76	Placebo	12	55	4.0	7.6	6.9/8.1	25.2	NR
Hanefeld [16]	444/111	Placebo	12	56	3.7	7.7	7.4/7.8	31.7	NR
Raz [47]	411/110	Placebo	18	55	4.6	8.0	7.7/8.2	32.1	31.8/32.3
Goldstein [23]	179/176	Placebo	24	53	4.5	8.7	8.2/8.9	31.9	NR
Rosenstock [35]	175/178	Placebo	24	56	6.1	8.1	7.2/7.8	31.5	32.6/31.5
Hermansen [24]	222/219	Placebo	24	56	8.7	8.3	7.8/8.6	31.0	31.5/31.2
Goldstein [23]	372/364	Placebo	54	53	4.4	8.8	7.1/7.8	32.2	NR
Charbonnel [48]	429/206	Placebo	24	54	6.3	8.0	7.3/7.9	31.3	NR
Aschner [49]	488/253	Placebo	24	54	4.4	8.0	7.3/8.2	30.5	30.3/30.5
Raz [50]	96/94	Placebo	30	55	8.0	9.2	8.3/9.1	30.2	NR
PN-040 [15]	352/178	Placebo	18	NR	NR	8.7	NR	NR	NR
PN-044 [15]	290/73	Placebo	12	NR	NR	7.6	NR	NR	NR

Saxagliptin
Rosenstock [51]	271/67	Placebo	12	53	1.0	7.9	7.1/7.7	31.0	30.7/30.7

ID/C: investigational drug/comparator; DM: diabetes mellitus; and glip.: glipizide.

aMean values.

Figure 2

Standardized differences (with 95% CI) of mean HbA1c at endpoint.

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………………………….

Safety: hypoglycemia

Information on hypoglycemia could be retrieved for 15 out of 18 trials with sitagliptin, and 19 out of 21 with vildagliptin. In some trials (1302 [14], 1303 [14], PN-035 [15], PN-036 [15], and PN-044 [15]), the overall number of hypoglycemic episodes was not reported. Of the remaining 34 studies, 5 (2 with sitagliptin and 3 with vildagliptin) reported that no hypoglycemic event had occurred during the trial. In the remaining studies, hypoglycemia was observed in 184 of 9944 patients treated with DPP-4 inhibitors (103 of 4573, 81 of 5100, and 0 of 271 with sitagliptin, vildagliptin and saxagliptin, respectively) and 293 of 5698 in comparator groups. The incidence of hypoglycemia with DPP-4 inhibitors (both sitagliptin and vildagliptin) was not significantly different from that observed with a placebo, even when those agents were used in combination with sulphonylureas or insulin (Fig. 3). In direct comparison, DPP-4 inhibitors were associated with a significantly lower hypoglycemic risk than sulphonylureas, whereas no significant differences were detected with respect to thiazolidinediones (Fig. 3). The number needed to harm (NNH) for sulphonylureas in comparison with DPP-4 inhibitors, with respect to hypoglycemia, on an yearly basis, was 128.

Figure 3

Mantel–Haenszel odds ratio (with 95% CI) for any hypoglycemia (logarithmic scale).

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All trials with DPP-4 inhibitors reported information on severe hypoglycemia, except 10 (6 with sitagliptin, 4 with vildagliptin). In three distinct trials [[16], [17], [18]], severe hypoglycemia was reported by 5 patients treated with sitagliptin, in monotherapy (N=4) or in combination with metformin (N=1), versus 9 cases in comparator groups (all treated with sulphonylureas). No case of severe hypoglycemia was reported in patients treated with vildagliptin, versus 5 with comparator (placebo in all cases); the severe hypoglycemic episodes detected in vildagliptin trials occurred in combination studies with sulphonylureas [19] or insulin [20].

……………………….

DPP-4 inhibitors have been proposed as an alternative to currently available therapies (sulphonylureas, thiazolidinediones or insulin), mainly as an add-on treatment in patients failing with metformin monotherapy. However, even the most recent version of the ADA–EASD consensus algorithm does not consider these drugs a viable option, except for selected cases [4]. The reasons for exclusion from the main treatment algorithm are scarce efficacy, limited amount of available evidence and high cost. With respect to available evidence, it should be recognized that several trials, which had not been included in previous meta-analyses [6], have been recently published [[19], [21], [22], [23], [24], [25]]. Furthermore, there are a relevant number of unpublished trials, the results of which have been disclosed on different websites, and are therefore available. The decision to publish a trial is, in most instances, performed by the sponsor which has a specific interest in pursuing the greater safety and tolerability of the new drug. This bias is unfortunate and limits the reliability of this and other meta-analysis, often based only on data provided from manufacturers; however, the retrieval of all available information should always be attempted, although the possibility of including some information of poorer methodological quality should be taken into account. The overall amount of evidence from randomized clinical trials which can be retrieved using this comprehensive approach is relevant, and probably sufficient for a reliable assessment of the clinical profile of this new class.

The overall efficacy on HbA1c of DPP-4 inhibitors in placebo-controlled trials is similar to that reported in previous meta-analyses [[1], [6], [7], [8]]. However, the greater number of available studies allowed separate analyses of trials in which DPP-4 inhibitors were used either as monotherapy or as an add-on to other agents. In fact, most currently available hypoglycemic treatments show a smaller additional effect on HbA1c when used as an add-on to metformin, in comparison with monotherapy trials [26]. Conversely, DPP-4 inhibitors produce a similar placebo-subtracted reduction of HbA1c either in monotherapy or as an add-on to other agents. This pattern resembles that of other drugs specifically active on post-prandial glucose, such as acarbose or glinides [26]. In fact, DPP-4 inhibitors, as well as GLP-1 receptor agonists, show a relevant effect on post-prandial hyperglycemia. Although data on post-prandial glucose measured through self-monitoring were not available, the results obtained in many trials with meal tests [[7], [27]] support the hypothesis of a specific action of DPP-4 inhibitors on post-prandial hyperglycemia.

Based on the considerations reported above, DPP-4 inhibitors, when used in combination with other drugs, should not be expected to be less effective on HbA1c than other agents (such as sulphonylureas, thiazolidinediones or insulin). Unfortunately, only a small number of head-to-head comparisons with other drugs are currently available. The efficacy of DPP-4 inhibitors on HbA1c, either in monotherapy or in combination with metformin, appears to be somewhat smaller than that of sulphonylureas, and similar to thiazolidinediones; the only two available comparisons with metformin, both in monotherapy, one with vildagliptin [28] and one with sitagliptin (PN-036 on www.merck.com/mrl/clinical_trials/results.html) suggest a smaller effect on HbA1c. It should be considered that most trials are of a relatively short duration and it is possible that sulphonylureas, which are known to produce a less durable effect on glucose than other available agents, [29] could provide less favorable results in the long-term.

Taken together, the present results on efficacy do not support the use of DPP-4 inhibitors in monotherapy as an alternative to metformin. On the other hand, these drugs appear to be effective as add-on treatments in patients failing with metformin monotherapy, with a specific effect on post-prandial glucose, although the short-term efficacy of sulphonylureas on HbA1c could be greater than that of DPP-4 inhibitors.

With respect to body mass index, this meta-analysis confirms the neutrality of DPP-4 inhibitors [[1], [6], [7], [8]]. In direct comparison, DPP-4 inhibitors appear to have an advantage in this respect over thiazolidinediones.

GLP-1 stimulates insulin secretion and inhibits glucagon production in a glucose-dependent manner, i.e. its effects are blunted when blood glucose reaches the lower limits of the normal range [30]. Therefore, DPP-4 inhibitors are expected to reduce glycemia with a low hypoglycemic risk. In fact, DPP-4 inhibitors do not induce any additional risk, in comparison with a placebo, either in monotherapy or in combination with sulphonylureas or insulin. This confirms the results of a recent meta-analysis performed on patient-level data from randomized clinical trials with sitagliptin [31]. Interestingly, in the only trial performed in insulin-treated patients, vildagliptin reduced the incidence of hypoglycemia in comparison with a placebo [20]. The mechanisms underlying this phenomenon need to be further elucidated. As expected, DPP-4 inhibitors do not increase the incidence of hypoglycemic episodes when compared with insulin-sensitizing drugs; on the other hand, they show a markedly reduced risk of hypoglycemia in head-to-head comparisons with sulphonylureas. This difference, which could be partly determined by a marginally greater efficacy of sulphonylureas on HbA1c, is consistent with the different mechanisms of action of the classes of drugs.

No patient experienced severe hypoglycemia during vildagliptin therapy. Unexpectedly, episodes of severe hypoglycemia occurred in five patients treated with sitagliptin, either in monotherapy or in combination with metformin, in three different trials [[16], [17], [18]]. Notably, two of those trials [[16], [18]], although published, did not report those events but since those trials were included in the registration data for drug approval in the US, the information on severe hypoglycemia can be retrieved from the FDA website. Furthermore, episodes of severe hypoglycemia were not considered in a recent meta-analysis of trials with sitagliptin, although a greater number of such events had occurred in comparator groups, which included sulphonylureas [31]. It should also be considered that some of the trials did not report any information on severe hypoglycemia, raising the possibility of a selective reporting bias. The occurrence of cases of severe hypoglycemia with DPP-4 inhibitor monotherapy is difficult to explain on the basis of the current knowledge of the mechanism of action of those drugs, and deserved further investigation.

Among other expected adverse events, the previously reported increased incidence of some infections during DPP-4 inhibitor therapy [[6], [8]] is confirmed, with sitagliptin, but not vildagliptin, associated with nasopharyngitis, and with a nonsignificant trend toward an increased risk of urinary tract infections. These results are consistent with those of a recent meta-analysis on patient-level data from trials with sitagliptin, which included only a fraction of the studies summarized in the present meta-analysis, and which showed a similar trend toward the increase of risk of nasopharyngitis with the DPP-4 inhibitor, although it failed to reach statistical significance [31]. It should be considered that DPP-4 is involved in the interaction between immune cells and that it could therefore modulate immune responses [32]; however, there is no evidence from mechanistic studies that inhibition of DPP-4 with currently available agents has an immunodepressant effect. Consistently, treatment with DPP-4 inhibitors does not appear to increase the risk of infections other than nasopharyngitis and urinary tract infections.

The introduction of a new class of drugs which are designed for long-term use always raises some concerns about safety during prolonged treatment. The possibility of rare, unexpected serious adverse events, which could not be detected in registration trials, should be considered. The number of reported deaths in available trials is still very small; however, there is no evidence suggesting an increase in mortality during treatment with DPP-4 inhibitors. The number of cardiovascular events registered in clinical trials is remarkably greater, although still inadequate to detect minor differences between groups. The two drugs which have been more thoroughly studied (sitagliptin and vildagliptin) do not seem to be associated with increased cardiovascular risk; in fact, the actual risk is lower than with comparators, although differences do not reach statistical significance. In fact, available data do not rule out the possibility of an increase of cardiovascular risk up to 28%, or of a reduction up to 54%. It should be considered that the duration of the available trials (up to one year) is insufficient to detect any effect of treatment (either detrimental or beneficial) on atherogenesis.

The addition of unpublished trials does not substantially modify the estimates of efficacy of DPP-4 inhibitors. However, the retrieval of unpublished, but publicly disclosed, information allowed the identification of some potentially interesting phenomena, such as cases of severe hypoglycemia with DPP-4 inhibitor monotherapy, which could not be detected in published papers.

The limitations of the present meta-analysis should be recognized and considered when interpreting the results. The analysis was performed on summary data, therefore lacking the accuracy of assessment which can be obtained when using patient-level data. For the very same reason, a time-to-event analysis for categorial outcomes (including cardiovascular events) could not be performed; the proportion of patients experiencing at least one event during the trial, which was used for meta-analysis, approximates the actual incidence of events only if this incidence is assumed to be constant throughout the duration of the trial. Furthermore, the number of subject studies and the duration of trials performed is insufficient to draw any definitive conclusion on the long-term cardiovascular safety of DPP-4 inhibitors.

In conclusion, DPP-4 inhibitors are effective in reducing HbA1c and post-prandial glucose; when used as an add-on to metformin, they show a medium-term efficacy on HbA1c similar to thiazolidinediones and marginally inferior to sulphonylureas, with a reassuring short- and medium-term safety profile. In fact, the hypoglycemic risk is low, and there is no evidence of detrimental effects on cardiovascular disease. In comparison with sulphonylureas or insulin, which have been proposed as first-choice agents in patients failing with metformin [4], DPP-4 inhibitors exhibit, at least in the short- and medium-term, a lower hypoglycemic risk and a more favorable action on body weight, at the price of a somewhat smaller efficacy and higher cost. The choice of the drugs to be used as add-ons to metformin in monotherapy failure largely depends on the relative weight attributed to each of these three components (safety, efficacy on HbA1c and cost).

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newly developed oxazolidinone antibiotics

Posted in Bacterial Resistance, Clinical & Translational, Commercialization, Curation, Disease Biology, Small Molecules in Development of Therapeutic Drugs, FDA Regulatory Affairs, Glycobiology: Biopharmaceutical Production, Infectious Disease & New Antibiotic Targets, Microbiology, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, tagged Antibacterial, gram positive bacteria, organic synthesis, oxazolidinones on November 27, 2015| Leave a Comment »

newly developed oxazolidinone antibiotics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

New Antibacterial oxazolidinones in pipeline by Wockhardt

by DR ANTHONY MELVIN CRASTO Ph.D

WCK ?

(5S)-N-{3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-1-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide

MF C₁₉ H₂₅ F₂ N₃ O_{5, MW 413.42}

Acetamide, N-[[(5S)-3-[3,5-difluoro-4-[4-hydroxy-4-(methoxymethyl)-1-piperidinyl]phenyl]-2-oxo-5-oxazolidinyl]methyl]-

CAS 957796-51-9

Antibacterial oxazolidinones

THIS MAY BE WCK 4086?????

PATENT

WO 2015173664, US8217058, WO 2012059823,

Oxazolidinone represent a novel chemical class of synthetic antimicrobial agents.Linezolid represents the first member of this class to be used clinically. Oxazolidinones display activity against important Gram-positive human and veterinary pathogens including Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococci (VRE) and β-lactam Resistant Streptococcus pneumoniae (PRSP). The oxazolidinones also show activity against Gram-negative aerobic bacteria, Gram-positive and Gram-negative anaerobes. (Diekema D J et al., Lancet 2001 ; 358: 1975-82).

Various oxazolidinones and their methods of preparation are disclosed in the literature. International Publication No. WO 1995/25106 discloses substituted piperidino phenyloxazolidinones and International Publication No. WO 1996/13502 discloses phenyloxazolidinones having a multisubstituted azetidinyl or pyrrolidinyl moiety. US Patent Publication No. 2004/0063954, International Publication Nos. WO 2004/007489 and WO 2004/007488 disclose piperidinyl phenyl oxazolidinones for antimicrobial use.

Pyrrolidinyl/piperidinyl phenyl oxazohdinone antibacterial agents are also described in Kim H Y et al., Bioorg. & Med. Chem. Lett., (2003), 13:2227-2230. International Publication No. WO 1996/35691 discloses spirocyclic and bicyclic diazinyl and carbazinyl oxazolidinone derivatives. Diazepeno phenyloxazolidinone derivatives are disclosed in the International Publication No. WO 1999/24428. International Publication No. WO 2002/06278 discloses substituted aminopiperidino phenyloxazolidinone derivatives.

Various other methods of preparation of oxazolidinones are reported in US Patent No. 7087784, US Patent No. 6740754, US Patent No. 4948801 , US Patent No. 3654298, US Patent No. 5837870, Canadian Patent No. 681830, J. Med. Chem., 32, 1673 (1989), Tetrahedron, 45, 1323 (1989), J. Med. Chem., 33, 2569 (1990), Tetrahedron Letters, 37, 7937-40 (1996) and Organic Process Research and Development, 11 , 739-741(2007).

Indian Patent Application No. 2534/MUM/2007 discloses a process for the preparation of substituted piperidino phenyloxazolidinones. International Publication No. WO2012/059823 further discloses the process for the preparation of phosphoric acid mono-(L-{4-[(5)-5-(acetylaminomethyl)-2-oxo-oxazolidin-3-yl]-2,6-difluorophenyl}4-methoxymethyl piperidine-4-yl)ester.

US Patent No. 8217058 discloses (5S)-N-{3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide as an antibacterial agent and its process for preparation.

PATENT

WO2015173664

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015173664&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

PATENT

http://www.google.st/patents/WO2007132314A2?cl=en

Wockhardt Ltd,

(3) (4)

PATENT

WO 2012059823

http://www.google.co.in/patents/WO2012059823A1?cl=en

Phosphoric acid mono-(l-{4-[(S)-5-(acetylamino- methyl)-2-oxo-oxazolidin-3-yl]-2,6-difluorophenyl}-4-methoxymethyl-piperidin-4-yl) ester of Formula (A),

the process comprising the steps of:

a) Converting intermediate of Formula (1) into intermediate of Formula (3)

b) Converting intermediate of Formula (3) into intermediate of Formula (5)

c) Converting intermediate of Formula (5) into intermediate of structure (6)

(5) <⁶> d) Converting intermediate of Formula (6) into intermediate of Formula (10)

e) Converting intermediate of Formula (10) into intermediate of Formula (11),

f) Converting intermediate of Formula (11) into compound of Formula (A) or Pharmaceutically acceptable salts thereof

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Rhodopsin role in ciliary trafficking

Posted in Blindness, Cargo, Cell Biology, Curation, Enzyme Induction, Gene Regulation, Genome Biology, Mutagenesis, Proteins, Proteomics, Signaling & Cell Circuits, Vision, tagged ciliary trafficking, GPCR phototransduction, guanine cyclase 1 (GC-1), guanylate cyclase isoforms, IFT, knockout mice, Mutagenesis, outer membrane, photoreceptor cells, phototransduction protein, retinal dystrophy, rhodopsin, rods and cones, signaling, trafficking pathways on November 27, 2015| Leave a Comment »

Rhodopsin role in ciliary trafficking

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>Jillian N Pearring,

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>William J Spencer,

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>Eric C Lieu,

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>Vadim Y Arshavsky
eLife 2015;10.7554/eLife.12058 http://dx.doi.org/10.7554/eLife.12058

Sensory cilia are populated by a select group of signaling proteins that detect environmental stimuli. How these molecules are delivered to the sensory cilium and whether they rely on one another for specific transport remains poorly understood. Here, we investigated whether the visual pigment, rhodopsin, is critical for delivering other signaling proteins to the sensory cilium of photoreceptor cells, the outer segment. Rhodopsin is the most abundant outer segment protein and its proper transport is essential for formation of this organelle, suggesting that such a dependency might exist. Indeed, we demonstrated that guanylate cyclase-1, producing the cGMP second messenger in photoreceptors, requires rhodopsin for intracellular stability and outer segment delivery. We elucidated this dependency by showing that guanylate cyclase-1 is a novel rhodopsin-binding protein. These findings expand rhodopsin’s role in vision from being a visual pigment and major outer segment building block to directing trafficking of another key signaling protein.

Photoreceptor cells transform information entering the eye as photons into patterns of neuronal electrical activity. This transformation takes place in the sensory cilium organelle, the outer segment. Outer segments are built from a relatively small set of structural and signaling proteins, including components of the classical GPCR phototransduction cascade. Such a distinct functional and morphological specialization allow outer segments to serve as a nearly unmatched model system for studying general principles of GPCR signaling (Arshavsky et al., 2002) and, in more recent years, a model for ciliary trafficking (Garcia-Gonzalo and Reiter, 2012; Nemet et al., 2015; Pearring et al., 2013; Schou et al., 2015; Wang and Deretic, 2014). Despite our deep understanding of visual signal transduction, little is known how the outer segment is populated by proteins performing this function. Indeed, nearly all mechanistic studies of outer segment protein trafficking were devoted to rhodopsin (Nemet et al., 2015; Wang and Deretic, 2014), which is a GPCR visual pigment comprising the majority of the outer segment membrane protein mass (Palczewski, 2006). The mechanisms responsible for outer segment delivery of other transmembrane proteins remain essentially unknown. Some of them contain short outer segment targeting signals, which can be identified through site-specific mutagenesis (Deretic et al., 1998; Li et al., 1996; Pearring et al., 2014; Salinas et al., 2013; Sung et al., 1994; Tam et al., 2000; Tam et al., 2004). A documented exception is retinal guanylate cyclase 1 (GC-1), whose exhaustive mutagenesis did not yield a distinct outer segment targeting motif (Karan et al., 2011).

GC-1 is a critical component of the phototransduction machinery responsible for synthesizing the second messenger, cGMP (Wen et al., 2014). GC-1 is the only guanylate cyclase isoform expressed in the outer segments of cones and the predominant isoform in rods (Baehr et al., 2007; Yang et al., 1999). GC-1 knockout in mice is characterized by severe degeneration of cones and abnormal light-response recovery kinetics in rods (Yang et al., 1999). Furthermore, a very large number of GC-1 mutations found in human patients cause one of the most severe forms of early onset retinal dystrophy, called Leber’s congenital amaurosis (Boye, 2014; Kitiratschky et al., 2008). Many of these mutations are located outside the catalytic site of GC-1, which raises great interest to understanding the mechanisms of its intracellular processing and trafficking.

In this study, we demonstrate that, rather than relying on its own targeting motif, GC-1 is transported to the outer segment in a complex with rhodopsin. We conducted a comprehensive screen of outer segment protein localization in rod photoreceptors of rhodopsin knockout (Rho-/- ) mice and found that GC-1 was the only protein severely affected by this knockout. We next showed that this unique property of GC-1 is explained by its interaction with rhodopsin, which likely initiates in the biosynthetic membranes and supports both intracellular stability and outer segment delivery of this enzyme. These findings explain how GC-1 reaches its specific intracellular destination and also expand the role of rhodopsin in supporting normal vision by showing that it guides trafficking of another key phototransduction protein.

GC-1 is the outer segment-resident protein severely down-regulated in rhodopsin knockout rods

GC-1 stability and trafficking require the transmembrane core of rhodopsin but not its outer 119 segment targeting domain

The findings reported in this study expand our understanding of how the photoreceptor’s sensory cilium is populated by its specific membrane proteins. We have found that rhodopsin serves as an interacting partner and a vehicle for ciliary delivery of a key phototransduction protein, GC-1. This previously unknown function adds to the well-established roles of rhodopsin as a GPCR visual pigment and a major building block of photoreceptor membranes. We further showed that GC-1 is unique in its reliance on rhodopsin, as the other nine proteins tested in this study were expressed in significant amounts and faithfully localized to rod outer segments in the absence of rhodopsin.

Our data consolidate a number of previously published observations, including a major puzzle related to GC-1: the lack of a distinct ciliary targeting motif encoded in its sequence. The shortest recombinant fragment of GC-1 which localized specifically to the outer segment was found to be very large and contain both transmembrane and cytoplasmic domains (Karan et al., 2011). Our study shows that GC-1 delivery requires rhodopsin and, therefore, can rely on specific targeting information encoded in the rhodopsin molecule. Interestingly, we also found that this information can be replaced by an alternative ciliary targeting sequence from a GPCR not endogenous to photoreceptors. This suggests that the functions of binding/stabilization of GC-1 and ciliary targeting are performed by different parts of the rhodopsin molecule. Our findings also shed new light on the report that both rhodopsin and GC-1 utilize intraflagellar transport (IFT) for their ciliary trafficking and co-precipitate with IFT proteins (Bhowmick et al., 2009). The authors hypothesized that GC-1 plays a primary role in assembling cargo for the IFT particle bound for ciliary delivery. Our data suggest that it is rhodopsin that drives this complex, at least in photoreceptor cells where these proteins are specifically expressed. Unlike GC-1’s reliance on rhodopsin for its intracellular stability or outer segment trafficking, rhodopsin does not require GC-1 as its expression level and localization remain normal in rods of GC-1 knockout mice ((Baehr et al., 2007) and this study). The outer segment trafficking of cone opsins is not affected by the lack of GC-1 either (Baehr et al., 2007; Karan et al., 2008), although GC-1 knockout cones undergo rapid degeneration, likely because they do not express GC-2 – an enzyme with redundant function. The primary role of rhodopsin in guiding GC-1 to the outer segment is further consistent with rhodopsin directly interacting with IFT20, a mobile component of the IFT complex responsible for recruiting IFT cargo at the Golgi network (Crouse et al., 2014; Keady et al., 2011).

It was also reported that GC-1 trafficking requires participation of chaperone proteins, most importantly DnaJB6 (Bhowmick et al., 2009). Our data suggest that GC-1 interaction with DnaJB6 is transient, most likely in route to the outer segment, since we were not able to co-precipitate DnaJB6 with GC-1 from whole retina lysates (Figure 5). In contrast, the majority of GC-1 co-precipitates with rhodopsin from these same lysates, suggesting that these proteins remain in a complex after being delivered to the outer segment. Although our data do not exclude that the mature GC-1-rhodopsin complex may contain additional protein component(s), our attempts to identify such components by mass spectrometry have not yielded potential candidates.

Interestingly, GC-1 was previously shown to stably express in cell culture where it localizes to either ciliary or intracellular membranes (Bhowmick et al., 2009; Peshenko et al., 2015). This strikes at the difference between the composition of cellular components supporting membrane protein stabilization and transport in cell culture models versus functional photoreceptors. The goal of future experiments is to determine whether these protein localization patterns would be affected by co-expressing GC-1 with rhodopsin, thereby gaining further insight into the underlying intracellular trafficking mechanisms.

Finally, GC-1 trafficking was reported to depend on the small protein, RD3, thought to stabilize both guanylate cyclase isoforms, GC-1 and GC-2, in biosynthetic membranes (Azadi et al., 2010; Zulliger et al., 2015). In the case of GC-1, this stabilization would be complementary to that by rhodopsin and potentially could take place at different stages of GC-1 maturation and trafficking in photoreceptors. Another proposed function of RD3 is to inhibit the activity of guanylate cyclase isoforms outside the outer segment in order to prevent undesirable cGMP synthesis in other cellular compartments (Peshenko et al., 2011a).

In summary, this study explains how GC-1 reaches its intracellular destination without containing a dedicated targeting motif, expands our understanding of the role of rhodopsin in photoreceptor biology and extends the diversity of signaling proteins found in GPCR complexes to a member of the guanylate cyclase family. Provided that the cilium is a critical site of GPCR signaling in numerous cell types (Schou et al., 2015), it would be interesting to learn whether other ciliary GPCRs share rhodopsin’s ability to stabilize and deliver fellow members of their signaling pathways