X-ray Diffraction of Water in Polyvinylpyrrolidone.

PVP is a hydrophilic polymer commonly used as an excipient in pharmaceutical formulations. Here we have performed time-resolved high-energy X-ray scattering experiments on pellets of PVP at different humidity conditions for 1-2 days. A two-phase exponential decay in water sorption is found with a peak in the differential pair distribution function at 2.85 Å, which is attributed to the average (hydrogen bonded) carbonyl oxygen-water oxygen distance. Additional scattering measurements on powders with fixed compositions ranging from 2 to 12.3 wt % H2O were modeled with Empirical Potential Structure Refinement (EPSR). The models reveal approximately linear relations between the carbonyl oxygen-water oxygen coordination number (nOC-OW) and the water oxygen-water oxygen coordination number (nOW-OW) versus water content in PVP. A stronger preference for water-water hydrogen bonding over carbonyl-water bonding is found. At all the concentrations studied the majority of water molecules were found to be randomly isolated, but a wide distribution of coordination environments of water molecules is found within the PVP polymer strands at the highest concentrations. Overall, the EPSR models indicate a continuous evolution in structure versus water content with nOW-OW=1 occurring at ∼12 wt % H2O, i.e., the composition where, on average, each water molecule is surrounded by one other water molecule.

Using containerless methods to develop amorphous pharmaceuticals.

BACKGROUND: Many pipeline drugs have low solubility in their crystalline state and require compounding in special dosage forms to increase bioavailability for oral administration. The use of amorphous formulations increases solubility and uptake of active pharmaceutical ingredients. These forms are rapidly gaining commercial importance for both pre-clinical and clinical use. METHODS: Synthesis of amorphous drugs was performed using an acoustic levitation containerless processing method and spray drying. The structure of the products was investigated using in-situ high energy X-ray diffraction. Selected solvents for processing drugs were investigated using acoustic levitation. The stability of amorphous samples was measured using X-ray diffraction. Samples processed using both spray drying and containerless synthesis were compared. RESULTS: We review methods for making amorphous pharmaceuticals and present data on materials made by containerless processing and spray drying. It was shown that containerless processing using acoustic levitation can be used to make phase-pure forms of drugs that are known to be difficult to amorphize. The stability and structure of the materials was investigated in the context of developing and making clinically useful formulations. CONCLUSIONS: Amorphous compounds are emerging as an important component of drug development and for the oral delivery of drugs with low solubility. Containerless techniques can be used to efficiently synthesize small quantities of pure amorphous forms that are potentially useful in pre-clinical trials and for use in the optimization of clinical products. GENERAL SIGNIFICANCE: Developing new pharmaceutical products is an essential enterprise to improve patient outcomes. The development and application of amorphous pharmaceuticals to increase absorption is rapidly gaining importance and it provides opportunities for breakthrough research on new drugs. There is an urgent need to solve problems associated with making formulations that are both stable and that provide high bioavailability. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Hydrogen Bonding in Amorphous Indomethacin.

Amorphous Indomethacin has enhanced bioavailability over its crystalline forms, yet amorphous forms can still possess a wide variety of structures. Here, Empirical Potential Structure Refinement (EPSR) has been used to provide accurate molecular models on the structure of five different amorphous Indomethacin samples, that are consistent with their high-energy X-ray diffraction patterns. It is found that the majority of molecules in amorphous Indomethacin are non-bonded or bonded to one neighboring molecule via a single hydrogen bond, in contrast to the doubly bonded dimers found in the crystalline state. The EPSR models further indicate a substantial variation in hydrogen bonding between different amorphous forms, leading to a diversity of chain structures not found in any known crystal structures. The majority of hydrogen bonds are associated with the carboxylic acid group, although a significant number of amide hydrogen bonding interactions are also found in the models. Evidence of some dipole-dipole interactions are also observed in the more structurally ordered models. The results are consistent with a distribution of Z-isomer intramolecular type conformations in the more disordered structures, that distort when stronger intermolecular hydrogen bonding occurs. The findings are supported by 1H and 2H NMR studies of the hydrogen bond dynamics in amorphous Indomethacin.

A High Energy X-ray Diffraction Study of Amorphous Indomethacin.

Amorphous pharmaceuticals often possess a wide range of molecular conformations and bonding arrangements. The x-ray pair distribution function (PDF) method is a powerful technique for the characterization of variations in both intra-molecular and inter-molecular packing arrangements. Here, the x-ray PDF of amorphous Indomethacin is shown to be particularly sensitive to the preferred orientations of the chlorobenzyl ring found in isomers in the crystalline state. In some cases, the chlorobenzyl ring has no preferred torsional angle in the amorphous form, while in others evidence of distinct isomer orientations are observed. Amorphous samples with no preferred torsion angles of the chlorobenzyl ring are found to favor enhanced inter-molecular hydrogen bonding, and this is reflected in the intensity of the first sharp diffraction peak. These significant variations in structure rule out amorphous Indomethacin as a possible standard for x-ray PDF measurements. At high humidity, time resolved PDF's for >40 h reveal water molecules forming hydrogen bonds with Indomethacin molecules. A simple linear hydrogen bond model indicates that water molecules in the wet amorphous form have similar hydrogen bond strengths to those found between Indomethacin dimers or chains in the dry amorphous form.

4-Aminopyridine derivatives enhance impulse conduction in guinea-pig spinal cord following traumatic injury.

4-Aminopyridine (4-AP), a potassium channel blocker, is capable of restoring conduction in the injured spinal cord. However, the maximal tolerated level of 4-AP in humans is 100 times lower than the optimal dose in in vitro animal studies due to its substantially negative side effects. As an initial step toward the goal of identifying alternative potassium channel blockers with a similar ability of enhancing conduction and with fewer side effects, we have synthesized structurally distinct pyridine-based blockers. Using isolated guinea-pig spinal cord white matter and a double sucrose gap recording device, we have found three pyridine derivatives, N-(4-pyridyl)-methyl carbamate (100 microM), N-(4-pyridyl)-ethyl carbamate (100 microM), and N-(4-pyridyl)-tertbutyl (10 microM) can significantly enhance conduction in spinal cord white matter following stretch. Similar to 4-AP, the derivatives did not preferentially enhance conduction based on axonal caliber. Unlike 4-AP, the derivatives did not change the overall electrical responsiveness of axons to multiple stimuli, indicating the axons recruited by the derivatives conducted in a manner similar to healthy axons. These results demonstrate the ability of novel constructs to serve as an alternative to 4-AP for the purpose of reversing conduction deficits.

Impermeant antitumor sulfonylurea conjugates that inhibit plasma membrane NADH oxidase and growth of HeLa cells in culture. Identification of binding proteins from sera of cancer patients.

The antitumor sulfonylurea LY237868 (N-(4-aminophenyl-sulfonyl)-N'-(4-chlorophenyl)urea) was conjugated through the A ring to alpha-cyclodextrin or agarose bead material (Affigel 10) to prepare impermeant conjugates for activity measurements and affinity isolation of binding proteins from serum. When conjugated to alpha-cyclodextrin, the resulting LY237868 conjugate inhibited both NADH oxidase activity and growth of HeLa cells in culture. The conjugate was at least one order of magnitude more potent as an inhibitor than the parent compound. These findings confirm previous results that demonstrate an antitumor sulfonylurea-binding protein with NADH oxidase activity at the external plasma membrane surface of HeLa cells that is shed into culture media conditioned by growth of HeLa cells. A comparable activity, responsive to sulfonylurea, was present in sera of cancer patients. LY237868 conjugated to agarose beads as the affinity support bound a large number of serum proteins. However, compared to serum from normal patients, the affinity support bound two proteins of M(r) approx. 33.5 and 29.5 not found in sera of normal patients. The 33.5 kDa protein from human sera reacted with antisera to a 33.5 kDa protein from culture media conditioned by growth of HeLa cells that blocked and immunoprecipitated the sulfonylurea-responsive activity from HeLa cell plasma membranes. The results point to the 33.5 kDa protein from cancer patient sera that bound to the sulfonylurea affinity support as representing the circulating equivalent of the previously identified 34 kDa sulfonylurea-binding protein, with NADH oxidase activity at the external cell surface of cultured HeLa cells and a corresponding 33.5 kDa protein shed into culture media conditioned by growth of HeLa cells.

Chemical reactivity in solid-state pharmaceuticals: formulation implications.

Solid-state reactions that occur in drug substances and formulations include solid-state phase transformations, dehydration/desolvation, and chemical reactions. Chemical reactivity is the focus of this chapter. Of particular interest are cases where the drug-substance may be unstable or react with excipients in the formulation. Water absorption can enhance molecular mobility of solids and lead to solid-state reactivity. Mobility can be measured using various methods including glass transition (T(g)) measurements, solid-state NMR, and X-ray crystallography. Solid-state reactions of drug substances can include oxidation, cyclization, hydrolysis, and deamidation. Oxidation studies of vitamin A, peptides (DL-Ala-DL-Met, N-formyl-Met-Leu-Phe methyl ester, and Met-enkaphalin acetate salt), and steroids (hydrocortisone and prednisolone derivatives) are discussed. Cyclization reactions of crystalline and amorphous angiotensin-converting enzyme (ACE) inhibitors (spirapril hydrochloride, quinapril hydrochloride, and moexipril) are presented which investigate mobility and chemical reactivity. Examples of drug-excipient interactions, such as transacylation, the Maillard browning reaction, and acid base reactions are discussed for a variety of compounds including aspirin, fluoxitine, and ibuprofen. Once solid-state reactions are understood in a pharmaceutical system, the necessary steps can be taken to prevent reactivity and improve the stability of drug substances and products.

Solution conformation of the thermolysin inhibitors carbobenzoxy-L-phenylalanine and beta-phenylpropionyl-L-phenylalanine and comparison of the solution conformation to the enzyme-bound conformation.

The conformations of enzyme inhibitors in solution and bound to the enzyme thermolysin are investigated as a convenient model for understanding the relationship between the conformation of drugs in solution and at the receptor. The solution conformations of carbobenzoxy-L-phenylalanine (I) and beta-phenylpropionyl-L-phenylalanine (II) are determined using nuclear magnetic resonance spectroscopy. These studies show that I and II have essentially the same conformation about both the alpha-beta bond and the C alpha-N bond in Me2SO-d6, acetone-d6, methanol-d4, chloroform-d, and D2O. In addition, the conformations of I and II are similar to phenylalanine and other phenylalanine derivatives. Comparison of the lowest energy solution conformer to that bound by the enzyme thermolysin shows that the lowest energy conformer (in solution) of carbobenzoxy-L-phenylalanine is bound by thermolysin; on the other hand, it is the highest energy conformer (in solution) of beta-phenylpropionyl-L-phenylalanine which is bound to the enzyme. This, to our knowledge, is one of the first experimental conformations of the hypothesis that an enzyme does not always bind the lowest energy conformer of an inhibitor.

Potential antitumor agents. Synthesis, reactivity, and cytoxicity of alpha-methylene carbonyl compounds.

The alpha-methylene lactones 9, 12, 21, and 24 were prepared by a mild, convenient alpha-methylenation process using the alpha-ethyloxalyl derivatives in nonoptimized yields ranging from 23 to 90%. The rates of reaction of these and several other lactones with cysteine at pH 7.4 and their KB toxicities were measured. These studies showed that neither the strained trans-fused alpha-methylene lactone 12 nor the hydroxy-alpha-methylene lactones 5 and 6 reacted with cysteine with rates comparable to elephantopin. Based on these limited studies, the rate of cysteine addition appears to be relatively insensitive to changes in strain energy and neighboring groups. In addition, the rate constant for reaction with cysteine did not correlate with cytotoxicity.

Properties of the nucleic acid photoaffinity labeling agent 3-azidoamsacrine.

This paper reports the study of the photochemical, physical, and biological properties of 3-azidoamsacrine. The binding of 3-azidoamsacrine to DNA was studied with UV spectroscopy. The UV spectral behavior is quite similar to that of the parent amsacrine and argues that 3-azidoamsacrine is a good photoaffinity labeling agent for amsacrine. The biological properties (cytotoxicity and mutagenicity) of 3-azidoamsacrine in the mammalian mutagenesis V79 and L5178Y assay systems were measured. Light-activated 3-azidoamsacrine is toxic, but not mutagenic, to V79 cells. 3-Azidoamsacrine with and without light activation, as well as amsacrine, are toxic and mutagenic to L5178Y cells. To probe the interactions of 3-azidoamsacrine with DNA, studies of the photoreactivity of this compound were conducted. 3-Azidoamsacrine was photolyzed in the presence of the plasmid pBR322, and the effect of the photoadducts on restriction endonuclease cleavage was investigated. Amsacrine and 3-azidoamsacrine, without light activation, did not block any of the restriction endonucleases. Light-activated 3-azidoamsacrine blocked cleavage by the restriction endonucleases AluI, HinfI, NciI, NaeI, DraI, Sau96I, HpaII, and HaeIII. Photolysis experiments with mononucleosides, blocked mononucleosides, dinucleotides, and DNA all indicated that 3-azidoamsacrine formed adducts with G and A. The structures of these adducts are discussed based upon mass spectral data. Thus, it appears that 3-azidoamsacrine covalently attaches to DNA and that this covalent binding results in the production of toxic and, in some cases, mutagenic lesions in mammalian cells and the inhibition of restriction endonuclease cleavage of DNA.

Comparison of solution conformational preferences for the hallucinogens bufotenin and psilocin using 360-MHz proton NMR spectroscopy.

The 360-MHz 1H NMR spectra of bufotenin and psilocin were obtained, both as the free bases in CDCl3 and as protonated salts in D2O. Coupling constants for the side-chain methylenes were derived using the LAOCN3 program. These time-averaged coupling constants indicate that the trans and gauche rotamers of both compounds have about equal energy in D2O. There is a slight excess of the trans rotamer of bufotenin in CDCl3. For psilocin, in contrast, the gauche form is highly favored in CDCl3. The magnitude of this stabilization was estimated at about 1 kcal/mol using rotamer populations and free energy of transfer from published partitioning studies. It is suggested that this could result from a very weak hydrogen bond. On the other hand, the difference in partitioning between bufotenin and psilocin, which seems to be a major determinant of biological activity, is largely due to a difference in the basicity of the two compounds. The pKa values for the amino group of psilocin and bufotenin were determined to be 8.47 and 9.67, respectively.

Effect of beta-alkyl substitution on D-1 dopamine agonist activity: absolute configuration of beta-methyldopamine.

beta-Methyldopamine and its enantiomers and racemic beta-phenyldopamine were synthesized and evaluated for dopamine D-1 agonist activity. In the dopamine-sensitive adenylate cyclase assay, beta-phenyldopamine had about one-sixth the activity of dopamine. Racemic beta-methyldopamine was less potent. The absolute configuration of beta-methyldopamine was determined to be R-(+) and S-(-). Evaluation of (R)-(+)- and (S)-(-)-beta-methyldopamine revealed no enantioselectivity for stimulation of adenylate cyclase.

Crystal structure of two crystal forms of 9 alpha-fluorocortisol acetate: variation of the conformation of the A ring of steroids due to crystal packing.

This paper reports the crystal structure of the propanol solvate of 9 alpha-fluorocortisol acetate, which crystallizes in the monoclinic space group P21 [a = 7.470 (6), b = 14.78 (1), c = 12.310 (9), beta = 105.2 degrees, Z = 2, R = 0.061, and the tetragonal unsolvated crystal form (a = b = 9.208 (2), c = 49.284 (9), P4(1)2(1)2, Z = 8, R = 0.050)]. The molecular structure of fluorocortisol acetate in the two crystal forms differs primarily in the A ring and acetoxy orientation. The A ring of the steroid in the tetragonal crystal was found to be disordered, and exists in both a normal and inverted conformation. The A ring of the steroid in the monoclinic propanol solvate has the normal (1 alpha, 2 beta half-chain) conformation. The differences in the conformation of the side chain and the A ring appear to reflect the conformational variability in 9 alpha-fluorocortisol acetate.

Crystal structure and solid-state behavior of aspirin anhydride crystals.

The crystal structure and solid-state behavior of aspirin anhydride were determined using single-crystal X-ray techniques and microscopic examination of the reacting crystals. The crystal structure and solid-state conformation of aspirin anhydride were similar to those of related compounds. The crystal packing of aspirin anhydride allows the initial product of the solid-state reaction to be predicted; however, this prediction could not be tested because the thermal degradation products reported in the literature appear to be those obtained from reaction in a liquid state.

Comparisons of rotamer populations of nialamide, azaperone, and chloroquine in solid state and in solution.

IR and NMR spectroscopy were combined with previously published X-ray crystallographic data to determine the solution conformations of the -(CH2)n- fragments of nialamide, azaperone, and chloroquine. The solution conformation of these compounds then was compared to the solid-state conformation. In addition, the limits of the IR-X-ray method are discussed. This paper shows that a combination of IR, NMR, and X-ray crystallographic data can lead to a complete picture of the conformations available to drugs. In addition, the danger of using solid-state conformational data alone to make pharmacological suggestions is illustrated.

Physical characteristics and chemical degradation of amorphous quinapril hydrochloride.

This study was designed to investigate the relationships between the solid-state chemical instability and physical characteristics of a model drug, quinapril hydrochloride (QHCl), in the amorphous state. Amorphous QHCl samples were prepared by rapid evaporation from dichloromethane solution and by grinding and subsequent heating of the crystalline form. Physical characteristics, including the glass transition temperature and molecular mobility, were determined using differential scanning calorimetry, thermogravimetric analysis, powder x-ray diffractometry, polarizing microscopy, scanning electron microscopy, and infrared spectroscopy. The amorphous form of QHCl, produced by both methods, has a T(g) of 91 degrees C. Isothermal degradation studies showed that cyclization of QHCl occurred at the same rate for amorphous samples prepared by the two methods. The activation energy was determined to be 30 to 35 kcal/mol. The rate of the reaction was shown to be affected by sample weight, dilution through mixing with another solid, and by altering the pressure above the sample. The temperature dependence for chemical reactivity below T(g) correlated very closely with the temperature dependence of molecular mobility. Above T(g), however, the reaction was considerably slower than predicted from molecular mobility. From an analysis of all data, it appears that agglomeration and sintering of particles caused by softening of the solid, particularly above T(g), and a resulting reduction of the particle surface/volume ratio play a major role in affecting the reaction rate by decreasing the rate of removal of the gaseous HCl product.

The interaction of granaticin with nucleic acids and pyruvate decarboxylase.

The interaction of granaticin with two different yeast ribonucleic acids, dialyzed transfer RNA and calf thymus DNA was studied. Contrary to previous reports, no binding of granaticin to DNA or RNA was observed. The visible spectrum of granaticin on direct mixing or equilibrium dialysis of granaticin with RNA or DNA was unchanged. Furthermore, granaticin did not displace acridine orange from DNA in competitive binding studies using fluorescence polarization. However, granaticin was shown to inhibit pyruvate decarboxylase. From the Ki for granaticin (3.8 mM) it was concluded that granaticin is as efficient as other 1,4-naphthoquinones in inhibiting pyruvate decarboxylase.

Analysis of solid-state Carbon-13 NMR spectra of polymorphs (benoxaprofen and nabilone) and pseudopolymorphs (cefazolin).

The solid-state 13C-NMR spectra of the polymorphs of benoxaprofen, nabilone, and cefazolin are reported using the cross-polarization/magic-angle spinning (CP/MAS) technique. In general, the spectra of the different crystal forms are different. In favorable cases the spectra of the drug in a pharmaceutical granulation can be discerned. These results provide a preliminary indication that solid-state NMR spectroscopy is a useful technique for the investigation of drug polymorphs and drugs in their dosage forms.

Degradation of crystalline ergocalciferol [vitamin D2, (3 beta,5Z,22E)-9,10-secoergosta-5,7,10(19),22-tetra en-3-ol].

The products of the degradation of crystalline ergocalciferol were investigated. These studies showed that numerous acidic and neutral oxidation products were formed resulting in the complete destruction of the triene functionality. Separation of the neutral products by preparative TLC led to material identified as the Windaus ketone IIa, 2,3,3a,4,5,6,7,7a beta-octahydro-7a alpha-methyl-1R-(1 alpha,1R,4R,5-trimethyl-2E-hexenyl)-4H-inden-4-one.

Hydration and dehydration of crystalline and amorphous forms of raffinose.

The trisaccharide raffinose was prepared in its crystal pentahydrate, anhydrous methanolate, and amorphous forms and evaluated with regard to dehydration and hydration properties at various temperatures and relative humidities. The pentahydrate, when stored at relative humidities (RHs) of < 60% but > 10%, showed no loss of water after 3 months of storage at 30 degrees C. When stored below 10% RH, only one water molecule could be removed over a period of 3 months, whereas within 24 h at 30 degrees C in a vacuum oven, two water molecules were removed with no change in crystal structure. Increasing the temperature to 60 degrees C progressively removed the remaining three molecules, causing the crystal, however, to collapse into an amorphous form identical to one prepared by lyophilization. Rehydration at 30 degrees C, which was sufficient to reduce the glass transition temperature to < 30 degrees C, rapidly restored the pentahydrate crystal structure. Rehydration of the methanolate also restored the pentahydrate structure. The significant amount of water accommodated by raffinose in both the crystalline and amorphous forms would appear to make it a potentially useful water scavenger in certain types of dosage forms.