Presence Of Various Excipients Research Articles

Prion-derived tetrapeptide stabilizes thermolabile insulin via conformational trapping

SummaryUnfolding followed by fibrillation of insulin even in the presence of various excipients grappled with restricted clinical application. Thus, there is an unmet need for better thermostable, nontoxic molecules to preserve bioactive insulin under varying physiochemical perturbations. In search of cross-amyloid inhibitors, prion-derived tetrapeptide library screening reveals a consensus V(X)YR motif for potential inhibition of insulin fibrillation. A tetrapeptide VYYR, isosequential to the β2-strand of prion, effectively suppresses heat- and storage-induced insulin fibrillation and maintains insulin in a thermostable bioactive form conferring adequate glycemic control in mouse models of diabetes and impedes insulin amyloidoma formation. Besides elucidating the critical insulin-IS1 interaction (R4 of IS1 to the N24 insulin B-chain) by nuclear magnetic resonance spectroscopy, we further demonstrated non-canonical dimer-mediated conformational trapping mechanism for insulin stabilization. In this study, structural characterization and preclinical validation introduce a class of tetrapeptide toward developing thermostable therapeutically relevant insulin formulations.

Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein–ligand binding and protein stability

A standard operating procedure for a fluorescence-based thermal shift assay (FTSA) is provided describing its typical applications, advantages and limitations. FTSA is a simple, robust, universal and quick assay to determine protein-ligand binding affinities and protein stabilities in the presence of various excipients and solution conditions. Therefore, the assay is very useful for the straightforward characterization of new recombinantly produced proteins. The assay has a wide dynamic range enabling simultaneous determination of affinities in the milimolar to picomolar range. The assay could be used for essentially any protein that is sufficiently soluble and stable in the studied aqueous solution. Here we provide examples and typical experimental protocols for both affinity and stability determinations.

Study of Degradation Kinetics of Magnesium Orotate Dihydrate by Spectroscopic Method

In the present work, we developed and validated five spectrophotometric methods for determination of magnesium orotate dihydrate in distilled water, pH 1.2, 4.5, 6.8, and 7.4 buffer systems. These media were selected to extend the applicability of the developed methods from the development of dosage forms to regular quality control. In all media, linear correlation was observed over the concentration range from 1 to 25 μg/mL. The developed methods were found to be selective and specific to the determination of magnesium orotate dihydrate in the presence of various excipients, as well as accurate, precise, and robust (the percent relative standard deviation was found to be less than 2%, and the percent recoveries were between 98 to 102%). The developed methods were also found to be highly sensitive, since the detection and quantification limits were found to be less than 1 μg/mL. The degradation kinetics studies reveal the stability of magnesium orotate dihydrate against hydrolysis (acidic or basic), oxidation, photolysis, and thermal treatment.

Optical properties of folic acid in phosphate buffer solutions: the influence of pH and UV irradiation on the UV-VIS absorption spectra and photoluminescence

Using UV-VIS absorption spectroscopy, photoluminescence (PL) and photoluminescence excitation (PLE), the photodegradation reactions of folic acid (FA) in phosphate buffer (PB) solutions were studied. Regardless of the PB solution’s pH, the UV-VIS spectra showed a gradual decrease in absorbance at 284 nm simultaneous with an increase in the absorbance of another band in the spectral range of 320–380 nm, which was downshifted under UV irradiation. The relative intensity of the FA PL band, situated in the spectral range 375–600 nm, was dependent on the pH of the PB solution. The FA PL intensity increased as increasing UV irradiation time up to 281 min. in PB solutions with pH values of 6.4 and 5.4. Under an emission wavelength of 500 nm, the position of the FA PLE spectrum changed as the PB solution pH varied from 7 to 5.4 and the irradiation time increased to 317 min. These changes were correlated with the formation of two photodegradation products, namely, pterine-6-carboxylic acid and p-amino-benzoyl-L-glutamic acid. According to UV-VIS spectroscopy and PL and PLE studies, the presence of various excipients in commercial pharmaceutical tablets does not affect the photodegradation of FA in PB solutions. Using IR spectroscopy, new evidences for the formation of the two photodegradation products of FA in PB solutions are shown.

Experimental Study on the Influence of Excipients on the Heterogeneous Crystallization and Dissolution Properties of an Active Pharmaceutical Ingredient

This study is based on the heterogeneous nucleation of active pharmaceutical ingredients (APIs) in the presence of various excipients widely used in the pharmaceutical industry. Carbamazepine (CBMZ) was successfully crystallized in the presence of the following heterosurfaces: α/β-lactose, β-d-mannitol, microcrystalline cellulose, and carboxymethyl cellulose. The successful crystallization of CBMZ FIII in the presence of all the excipients was confirmed by powder X-ray diffraction and scanning electron microscopy, while CBMZ crystals’ apposition was confirmed using in situ scanning electronic microscopy-Raman. A pronounced improvement in the dissolution of CBMZ FIII was observed when crystallized in the presence of excipients when compared with CBMZ FIII recrystallized using the same conditions in the absence of the excipients. The isolated solids could be simply tabletted by direct compression upon mixing with the desired amount of disintegrant and lubricant. Hence employing this process could potentiall...

In vivo controlled release of fenretinide from long-acting release depots for chemoprevention of oral squamous cell carcinoma recurrence

Local, long-acting release fenretinide (4HPR) millicylindrical implants were prepared and evaluated for their release kinetics in vivo and their ability to suppress oral cancer tumor explant growth. Poly(lactic-co-glycolic acid)(PLGA) implants were prepared as a function of drug loading and the presence of various excipients (pore-formers, solubilizers, crystallization inhibitors) to enhance release of the insoluble 4HPR. Release kinetics and bioerosion of PLGA were monitored both in vitro in a PBS/Tween 80 buffer and in vivo by recovery of the drug remaining at the injection site. 4HPR was released from PLGA implants much slower in vivo than in the drug solubilizing media in vitro, with a 3-week lag phase and continuous release of >2 months, but showed some release enhancement by addition of solubilizers. Water-soluble PVA/sucrose implants for release of 4HPR served to determine if drug dissolution provided suitable controlled release without the PLGA, and this formulation showed continuous drug release over 6 weeks in vivo. Placement of PLGA-4HPR implants adjacent to oral cancer tumor murine xenografts showed inhibition of tumor growth relative to sham implants, indicating the potential for the local 4HPR delivery approach to be useful for oral cancer chemoprevention.

Elucidating the weak protein-protein interaction mechanisms behind the liquid-liquid phase separation of a mAb solution by different types of additives

Liquid-liquid phase separation (LLPS) has long been observed during the physical stability investigation of therapeutic protein formulations. The buffer conditions and the presence of various excipients are thought to play important roles in the formulation development of monoclonal antibodies (mAbs). In this study, the effects of several small-molecule excipients (histidine, alanine, glycine, sodium phosphate, sodium chloride, sorbitol and sucrose) with diverse physical-chemical properties on LLPS of a model IgG1 (JM2) solutions were investigated by multiple techniques, including UV–vis spectroscopy, circular dichroism, differential scanning calorimetry/fluorimetry, size exclusion chromatography and dynamic light scattering. The LLPS of JM2 was confirmed to be a thermodynamic equilibrium process with no structural changes or irreversible aggregation of proteins. Phase diagrams of various JM2 formulations were constructed, suggesting that the phase behavior of JM2 was dependent on the solution pH, ionic strength and the presence of other excipients such as glycine, alanine, sorbitol and sucrose. Furthermore, we demonstrated that for this mAb, the interaction parameter (kD) determined at low protein concentration appeared to be a good predictor for the occurrence of LLPS at high concentration.

Abstract 4482: Development of localized cisplatin chemotherapy: from benchside to investigational new drug application

Abstract Many cancers preferentially spread to the lymphatics before systemic dissemination. The purpose of this study is to develop intralymphatically-targeted cisplatin nanoparticles for treating locally advanced head and neck cancer. This treatment methodology may significantly reduce the side effects and increase the efficacy of cisplatin-based chemotherapy in both humans and companion animals, compared to conventional intravenous chemotherapies. Cisplatin was chemically conjugated to hyaluronan (HA) to form hyaluronan-cisplatin (HA-Pt) nanoparticles. The nanoparticles were purified and concentrated using a tangential flow filtration system. The cisplatin substitution degree, particle size, molecular weight, viscosity, osmolality, sterility, bioburden, particulate content, impurity profiles, and stability of the formulation in the presence of various excipients were determined. All analytical methods utilized were fully validated according to FDA and/or USP guidelines. The pharmacokinetics, biodistribution and safety of subcutaneously injected HA-Pt were evaluated in animals. Anti-cancer efficacy of HA-Pt was determined in mice bearing cisplatin sensitive xenografts (1/wk for 3wks; 3.5mg/kg). Canines with spontaneously-occurring limb lymphomas were given a single 0.85-mg/kg subcutaneous dose peritumorally. Canines with spontaneously-occurring oral squamous cell carcinoma, oral melanoma, or nasal tumors received 3 doses at 10 to 15 mg/m2, peritumorally. The cisplatin conjugation degree on HA was 15-20 w/w%. HA-Pt demonstrated antiproliferative efficacy similar to standard cisplatin in vitro with sustained release of cisplatin (t1/2=10h). The characteristics of HA-Pt meet FDA requirements for injectable formulations. HA-Pt resulted in higher plasma area-under-the-curve (AUC) and lower Cmax in rats. Murine xenografts demonstrated improved complete and partial responses to HA-Pt treatment compared to standard cisplatin therapy. The HA-Pt reduced Cmax 5.5 fold and increased the canine plasma AUC of cisplatin 5.4 fold and the lymph node concentration 18 fold compared to i.v. cisplatin. The tumor:plasma ratio in canines was 429, indicating the nanoparticles concentrated drug within the tumor. A single injection of HA-Pt resulted in size reduction of the metastatic lymph nodes of canine oral melanoma. After the completion of 3 treatments, the canine oral tumor was completely eradicated (no recurrence within a year) and swelling was greatly reduced. Stable disease was observed for most of the dogs. This study demonstrates that subcutaneous delivery of HA-Pt formulations may be a promising treatment regimen to improve tumor drug accumulation and therapeutic efficacy. The successful completion of a series of GLP rodent and canine study will warrant a submission of an Investigational New Drug Application to the FDA. Citation Format: Shuang Cai, Jeffrey Bryan, Daniel Aries, Laird Forrest. Development of localized cisplatin chemotherapy: from benchside to investigational new drug application. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2014-4482

Degradation Kinetics and Mechanism of an Oxadiazole Derivative, Design of a Stable Drug Product for BMS-708163, a γ-Secretase Inhibitor Drug Candidate

The stability of a 1,2,4-oxadiazole derivative, BMS-708163, A, was studied in the cosolvent mixture of acetonitrile-water at various pH values, in the solid state and in the presence of various excipients. The objective of this study was to develop a deep understanding of the stability of compound A based on its degradation kinetics and mechanism to enable design of a robust drug product. A series of isotopically (13) C- and (15) N-labeled compounds were synthesized and their degradation was studied by liquid chromatography-mass spectrometry and nuclear magnetic resonance to prove the degradation mechanism. Compound A exhibited maximum stability at a pH range of 3-5. In forced degradation studies, higher or lower pH resulted in an increase in degradation rate. At low pH, the N-4 atom on the 1,2,4-oxadiazole ring is protonated, followed by nucleophilic attack on the activated methine carbon to cause ring opening to form aryl nitrile degradation product, compound B. At high pH, the nucleophilic attack occurs on the methine carbon to generate an anion on N-4. Subsequent proton capture from a proton donor, such as ambient water, facilitates ring opening to generate compound B. In the absence of a proton donor, such as in dry acetonitrile, anion on N-4 will go back to compound A. Therefore, compound A is stable in absence of proton donor. This study defines the package condition and microenvironmental pH under which compound A can be formulated into a stable product.

The effect of hydroxypropyl-beta-cyclodextrin and sucrose on the sublingual absorption of midazolam in rabbits

Sublingually administered midazolam is commonly used for premedication of pediatric patients. However, the irritating taste and low aqueous solubility of midazolam complicate its sublingual use. Cyclodextrin complexation can be used to improve both the taste and aqueous solubility of drugs, but based on earlier studies, the complexation efficiency of midazolam is relatively low. In the present study, the complexation of midazolam with hydroxypropyl-beta-cyclodextrin (HP-β-CD) was investigated in the presence of various excipients. The aqueous solubility of midazolam improved significantly when HP-β-CD was used together with sucrose. Sucrose alone did not increase the solubility of midazolam. In addition, sucrose increased the apparent stability constant of the midazolam/HP-β-CD complex. The pharmacokinetics of midazolam in different dosage forms was investigated in rabbits (dose 1mg/rabbit) after intravenous administration of midazolam solution and after sublingual administration of midazolam solution, midazolam/HP-β-CD/sucrose solution or midazolam/HP-β-CD/sucrose powder. Midazolam displayed rapid sublingual absorption (mean tmax⩽30min from the liquid formulations and 60min from the solid formulation) with high absolute bioavailability (>68%) from all evaluated formulations. Based on the results, HP-β-CD and sucrose can be utilized together to prepare more concentrated and palatable midazolam formulations for sublingual administration in pediatric patients.

A fast and efficient determination of amines and preservatives in cough and cold liquid and suspension formulations using a single isocratic ion-pairing high power liquid chromatography method

A single, highly selective ion-pairing reverse phase-high power liquid chromatography (RP-HPLC) method has been developed for the determination of amines and preservatives in a wide range of Tylenol((R)) liquid and suspension liquid products. As with many OTC products, the challenge is to quantitatively extract the analytes from difficult matrices and specifically analyze them in the presence of various excipients and flavors. Historically, separate analytical methods were used for each class of analytes (acids, bases and neutral compounds). In this method a mobile phase consisting of a buffered ion-pairing agent with acetonitrile, methanol and tetrahydrofuran was used to separate the charged amines from neutral and acidic compounds on a Phenomenex LUNA C8(2) 75 x 4.6 mm i.d. analytical column with a 3-microm particle size. The analytes include acids (benzoic acid), bases (pseudoephedrine, chlorpheniramine, dextromethorphan, doxylamine and diphenhydramine) and a neutral compound (butylparaben). The effects of pH, the chain length of the ion-pairing reagent, ionic strength and organic modifiers on the separation are discussed. The method is linear from 15 to 150% of the target amounts. The optimized method proves to be specific, robust and accurate for the analysis of the compounds.

The Secondary Structure and Aggregation of Lyophilized Tetanus Toxoid

Tetanus toxoid (TT), the vaccine for tetanus, is an important protein antigen and candidate for sustained release from polymeric matrices. During administration from the latter, the solid (e.g., lyophilized) protein will be exposed to elevated levels of temperature and moisture, conditions which trigger its aggregation. To examine the connection between this aggregation and the structure of the TT molecule in the solid state, Fourier-transform infrared (FTIR) spectroscopy was employed to determine the secondary structure of TT in the presence of various excipients. We found that excipient-free TT undergoes a significant alteration (mostly reversible) in the secondary structure during lyophilization. Specifically, more than half the total α-helix content was lost with a concomitant increase in β-sheet structure. The extent of structural alterations in the presence of 1:5 (g:g protein) NaCl, sorbitol, or poly-(ethylene glycol), did not correlate with stability conferred towards moisture-induced aggregation. These results suggest that the degree of retention of the native protein structure in the dry state is not a general predictor of stability for the “wetted” solid within polymer controlled-release vehicles.