Electronic and resonance effects on the ionization of structural analogues of efavirenz.

The solubility of 4 analogues of efavirenz was studied as a function of pH. The study evaluated the ionization behavior and determined the relative contribution of electronegative substituents versus resonance effects on the pK(a) value of the cyclic carbamate. The most profound lowering effect on the pK(a) was due to the presence of multiple electronegative substituents and in particular the trifluoromethyl and acetylene groups. The presence of chlorine on the benzoxazinone ring was found to have a slight impact on the pK(a), although to a lesser extent. In the absence of any functional groups on the benzoxazinone ring system, the pKa shifted to a value of 13.2, which is 3 pH units above that of efavirenz and more closely correlates with typical literature values for cyclic carbamates.

Degradation of a fluoropyridinyl drug in capsule formulation: degradant identification, proposed degradation mechanism, and formulation optimization.

The purpose of this paper was to investigate the degradation chemistry of a fluoropyridinyl drug candidate in capsule formulation and to optimize the formulation based on a proposed degradation mechanism. Small developmental batches of capsules were made by tituration of drug substance and excipients using a mortar and pestle, followed by manual encapsulation. Degradants were identified by LC-MS/MS and LC-photodiode array detector (PDA) and were monitored by LC-ultraviolet detector (UVD) during stability studies. It was found that the drug could undergo a nucleophilic substitution reaction in which hydroxyl groups replace the fluorine substituents on the pyridine rings. The initial degradation rate is independent of the drug concentration but dependent on the temperature, the pH of the microenvironment, and the excipient type. On the basis of these experimental results, a nucleophilic substitution reaction mechanism for the degradation was proposed and a successful capsule formulation was developed.

Expanded-spectrum nonnucleoside reverse transcriptase inhibitors inhibit clinically relevant mutant variants of human immunodeficiency virus type 1.

A research program targeted toward the identification of expanded-spectrum nonnucleoside reverse transcriptase inhibitors which possess increased potency toward K103N-containing mutant human immunodeficiency virus (HIV) and which maintain pharmacokinetics consistent with once-a-day dosing has resulted in the identification of the 4-cyclopropylalkynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 961 and DPC 963 and the 4-cyclopropylalkenyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 082 and DPC 083 for clinical development. DPC 961, DPC 963, DPC 082, and DPC 083 all exhibit low-nanomolar potency toward wild-type virus, K103N and L100I single-mutation variants, and many multiply amino acid-substituted HIV type 1 mutants. This high degree of potency is combined with a high degree of oral bioavailability, as demonstrated in rhesus monkeys and chimpanzees, and with plasma serum protein binding that can result in significant free levels of drug.

Orally active isoxazoline glycoprotein IIb/IIIa antagonists with extended duration of action.

Modification of the alpha-carbamate substituent of isoxazoline GPIIb/IIIa (alphaIIb beta3) antagonist DMP 754 (7) led to a series of alpha-sulfonamide and alpha-sulfamide diaminopropionate isoxazolinylacetamides which were found to be potent inhibitors of in vitro platelet aggregation. Aryl- and heteroaryl-alpha-sulfonamide groups, in conjunction with (5R)-isoxazoline (2S)-diaminopropionate stereochemistry, were found to impart a pronounced duration of antiplatelet effect in dogs, potentially due to high affinity for unactivated platelets. Isoxazolylsulfonamide 34b (DMP 802), a highly selective GPIIb/IIIa antagonist, demonstrated a prolonged duration of action after iv and po dosing and high affinity for resting and activated platelets. The prolonged antiplatelet profile of DMP 802 in dogs and the high affinity of DMP 802 for human platelets may be predictive of clinical utility as a once-daily antiplatelet agent.

Applications of modulated differential scanning calorimetry in preformulation studies.

Characterization of the thermal properties of active pharmaceutical ingredients is critical in the selection of appropriate physical forms for development and defining proper manufacturing, handling and storage conditions of those chemical entities. Modulated differential scanning calorimetry (MDSC) has proven to be an effective tool in the thorough characterization of thermal behavior of compounds in preformulation studies. Selected applications of MDSC for various preclinical compounds are presented, thereby demonstrating the utility of this analytical method in the determination of glass transitions, characterization of desolvation and degradation processes as well as in the study of polymorphic transformations and crystallizations.

Determination of the pKa and pH-solubility behavior of an ionizable cyclic carbamate, (S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4- (trifluoromethyl)-2H-3,1-benzoxazin-2-one (DMP 266).

The solubility of a nonnucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl )- 2H-3,1-benzoxazin-2-one (DMP 266), was investigated as a function of pH. A dramatic increase in the aqueous solubility was observed at pH > or = 10, which was consistent with going from a neutral to a charged species. The ionization of the proton positioned on the carbamate functionality was confirmed spectrophotometrically (pKa = 10.1). The spectrophotometric result was in excellent agreement with that obtained from the solubility studies (pKa = 10.2). The ionization behavior of DMP 266 represents a unique case in which the pKa for a carbamate functional group is quite low. The anomalous pKa value may be attributed to stabilization of the negatively charged species through inductive effects, which originate from the surrounding substituents and delocalization of the negative charge via resonance effects.

Determination of intracellular levels of 6-mercaptopurine metabolites in erythrocytes utilizing capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis proved to be a useful technique for the analysis of intracellular levels of 6-thioguanosine mono-, di-, and triphosphate with analysis times of 20 min. Conditions required for baseline separation of the thioguanine nucleotides consisted of a 25 mM KH2PO4 (pH 8.0) buffer and a separation voltage of +28 kV. Laser-induced fluorescence detection (lambda ex = 325 nm, lambda em = 410 nm) of the thioguanine nucleotide metabolites of 6-mercaptopurine (6-MP) was possible following oxidation of the thiol functionality. Tedious extraction procedures involving mercury cellulose resins or phenyl mercury adduct formation, which had been required previously for the selective extraction of thiopurines from erythrocytes, were unnecessary due to the overall specificity of the approach. However, the inclusion of 50 mM EDTA in the sample preparation was required to inhibit the anabolic/catabolic enzymatic activity, which was responsible for the degradation of the analytes. The method demonstrated linearity from 5 to 1700 pmol/100 microliters red blood cells for the three analytes (RSDs < or = 8%). The feasibility of the method was demonstrated for the quantitation of 6-thioguanine nucleotides in patients receiving either oral or intravenous 6-MP therapy.

Improvements in detection sensitivity for the determination of ivermectin in plasma using chromatographic techniques and laser-induced fluorescence detection with automated derivatization.

Improvement in detection sensitivity for the analysis of ivermectin was observed through utilization of laser-induced fluorescence detection and by manipulation of chromatographic conditions. Gradient elution used in combination with narrow-bore chromatography and conventional fluorescence detection resulted in a limit of quantitation for the major homologue of ivermectin of 0.01 ng/ml in dog plasma. Laser-induced fluorescence detection with isocratic chromatographic conditions also resulted in a limit of quantitation of 0.01 ng/ml in dog plasma, which is a six-fold improvement over previously reported methods. Introduction of an automated procedure for the derivatization and injection of samples reduced the amount of sample handling, eliminated the potential for analyte/internal standard degradation and contributed to the overall ease of analysis.

Applications of capillary electrophoresis in pharmaceutical analysis.

The role of capillary electrophoresis (CE) in the analysis of peptide/proteins, chiral pharmaceuticals, and other small-molecule drugs has been reviewed. Potential uses of CE range from purity and structural confirmation to a micropreparative technique. Strategies for the prevention of protein wall adsorption include the use of extreme pH values, surface-modified capillaries, and high ionic strengths employing salts of alkali metals or by the addition of zwitterionic surfactants to the background electrolyte. Chiral separations of amino acids and other racemic pharmaceuticals have been achieved by micellar electrokinetic chromatography or by the introduction of cyclodextrins/modified cyclodextrins or other reagents to the running buffer. Applications of capillary electrophoresis to the analysis of small-molecule pharmaceuticals include determinations of drugs and/or excipients in various pharmaceutical preparations and the analysis of miscellaneous pharmaceuticals in standard solutions and biological fluids. The complementary nature of capillary electrophoresis and HPLC, in addition to future expectations of CE in pharmaceutical analysis, is discussed.