Preformulation study of NSC-726796.

A stability-indicating high-performance liquid chromatography method to quantify 2-(2,4-difluorophenyl)-4,5,6,7-tetrafluoroisoindoline-1,3-dione (NSC-726796) and its three main degradation products was developed. This method was used to investigate its degradation kinetics and mechanism. The reaction follows first-order kinetics and appears to be base catalyzed with the maximum stability at pH 1. The products were identified as 2-(2,4-difluorophenylcarbamoyl)-3,4,5,6-tetrafluorobenzoic acid (NSC-749820), 2,4-difluoroaniline, and tetrafluorophthalic acid. The parent drug, NSC-726796, was also found to react with methanol and ethanol. NSC-726796 demonstrates antiangiogenic activity, however, when its degradant NSC749820 does not show antiangiogenic activity.

Stability of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in combination.

In vivo, the DNA methyltransferase inhibitor, 5-fluoro-2'-deoxycytidine (FdCyd, NSC-48006), is rapidly converted to its unwanted metabolites. Tetrahydrouridine (THU, NSC-112907), a cytidine deaminase inhibitor can block the first metabolic step in FdCyd catabolism. Clinical studies have shown that co-administration with THU can inhibit the metabolism of FdCyd. The National Cancer Institute is particularly interested in a 1:5 FdCyd/THU formulation. The purpose of this study was to investigate the in vitro pH stability of FdCyd and THU individually and in combination. A stability-indicating high-performance liquid chromatography method for the quantification of both compounds and their degradants was developed using a ZIC(R)-HILIC column. The effect of THU and FdCyd on the in vitro degradation of each other was studied as a function of pH from 1.0 to 7.4 in aqueous solutions at 37 degrees C. The degradation of FdCyd appears to be first-order and acid-catalyzed. THU equilibrates with at least one of its degradants. The combination of FdCyd and THU in solution does not affect the stability of either compound. The stability and compatibility of FdCyd and THU in the solid state at increased relative humidity and at various temperatures are also evaluated.

Preformulation and pharmacokinetic studies on antalarmin: a novel stress inhibitor.

The preformulation, solubilization and pharmacokinetic evaluation of antalarmin, a stress inhibitor, have been conducted. Antalarmin has a poor water solubility of less than 1 microg/mL and is weakly basic with an experimentally determined pK(a) of 5.0. Multiple solubilization approaches including pH-control either alone or in combination with cosolvents, surfactants and complexing agents have been investigated. The applicability of lipid-based systems has also been explored. Four formulations, each with a targeted drug loading capacity of 100 mg/mL, show potential for oral administration. Three of these formulations are aqueous solutions (10% ethanol + 40% propylene glycol; 20% cremophor EL; 20% sulfobutylether-beta-cyclodextrin) each buffered at pH 1. The fourth formulation is a lipid-based formulation comprising of 20% oleic acid, 40% cremophor EL and 40% Labrasol. No precipitation was observed following dilution of the four formulations with water and enzyme free simulated gastric fluid. However, only the lipid-based formulation successfully resisted drug precipitation following dilution with enzyme free simulated intestinal fluid. Pharmacokinetic analysis conducted in rats revealed that the 20% cremophor EL solution formulation has a fivefold higher oral bioavailability compared to a suspension formulation. The lipid-based formulation resulted in over 12-fold higher bioavailability as compared to the suspension formulation, the highest amongst the formulations examined.

Solubilization of two structurally related anticancer drugs: XK-469 and PPA.

The efficiency of a solubilization technique is determined by the physical-chemical properties of the drug. This study investigates the solubilization on two structurally related anticancer drugs, XK-469 and PPA. XK-469 is much less polar than PPA with an intrinsic solubility of 0.000274 mg/mL, which is about 10,000 fold less than that of PPA. Fortunately, its physical-chemical properties make it much more formulatable. An ionizable drug can be solubilized by pH adjustment with cosolvency, micellization, or complexation. Both XK-469 and PPA are weak acids with pKa values of 2.7 and 2.9, respectively. Thus, they can be solubilized by pH adjustment. At pH 4.55, neither cosolvency, micellization nor complexation has much effect on the solubility of PPA. However, these techniques can significantly increase the solubility of XK-469. In fact, the solubility of XK-469 in 20% HPbetaCD at pH 4.55 is 5.85 mg/mL, which is more than 20,000 times greater than its intrinsic solubility. With the solubilization descriptors obtained from the experimental data for both unionized and ionized drug species at pH 1.0 and pH 4.55, the solubility of each drug at any pH and excipient concentration can be estimated. Then, a solubilization technique can be chosen for preparing a desired final drug concentration.

Influence of the structure of drug moieties on the in vitro efficacy of HPMA copolymer-geldanamycin derivative conjugates.

PURPOSE: To optimize the structure of geldanamycin (GDM) derivative moieties attached to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via an enzymatically degradable spacer. METHODS: HPMA copolymers containing different AR-GDM (AR = 3-aminopropyl (AP), 6-aminohexyl (AH), and 3-amino-2-hydroxypropyl AP(OH)) were synthesized and characterized. Their cytotoxicity towards the A2780 human ovarian carcinoma cells was evaluated. RESULTS: The cytotoxic efficacy of HPMA copolymer-AR-GDM conjugates depended on the structure of AR-GDM. Particularly, HPMA copolymer-bound AH-GDM, which possessed the longest substituent at the 17-position, demonstrated the highest efficacy among the polymer-bound GDM derivatives; however the activity of free AH-GDM was lower than that of the other free AR-GDMs. The relative increase of the activity of macromolecular AH-GDM when compared to AP-GDM or AP(OH)-GDM correlated with the enhanced recognition of AH-GDM terminated oligopeptide side-chains by the active site of the lysosomal enzyme, cathepsin B. Drug stability and further stabilization upon binding to HPMA copolymer also contributed to the observed phenomena. CONCLUSIONS: AH-GDM was found to be a suitable GDM derivative for the design of a drug delivery system based on HPMA copolymers and enzymatically-degradable spacers.

Identification and antimicrobial susceptibility of Alcaligenes xylosoxidans isolated from patients with cystic fibrosis.

In the past decade, potential pathogens, including Alcaligenes species, have been increasingly recovered from cystic fibrosis (CF) patients. Accurate identification of multiply antibiotic-resistant gram-negative bacilli is critical to understanding the epidemiology and clinical implications of emerging pathogens in CF. We examined the frequency of correct identification of Alcaligenes spp. by microbiology laboratories affiliated with American CF patient care centers. Selective media, an exotoxin A probe for Pseudomonas aeruginosa, and a commercial identification assay, API 20 NE, were used for identification. The activity of antimicrobial agents against these clinical isolates was determined. A total of 106 strains from 78 patients from 49 CF centers in 22 states were studied. Most (89%) were correctly identified by the referring laboratories as Alcaligenes xylosoxidans. However, 12 (11%) strains were misidentified; these were found to be P. aeruginosa (n = 10), Stenotrophomonas maltophilia (n = 1), and Burkholderia cepacia (n = 1). Minocycline, imipenem, meropenem, piperacillin, and piperacillin-tazobactam were the most active since 51, 59, 51, 50, and 55% of strains, respectively, were inhibited. High concentrations of colistin (100 and 200 microg/ml) inhibited 92% of strains. Chloramphenicol paired with minocycline and ciprofloxacin paired with either imipenem or meropenem were the most active combinations and inhibited 40 and 32%, respectively, of strains. Selective media and biochemical identification proved to be useful strategies for distinguishing A. xylosoxidans from other CF pathogens. Standards for processing CF specimens should be developed, and the optimal method for antimicrobial susceptibility testing of A. xylosoxidans should be determined.

Cathelicidin peptides inhibit multiply antibiotic-resistant pathogens from patients with cystic fibrosis.

Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid P. aeruginosa strains (MIC, 0.06 to 8 microg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml). Peptides had modest activities against S. maltophilia and A. xylosoxidans (MIC range, 1.0 to > 32 microg/ml), but none inhibited B. cepacia. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P. aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.

Use of pure t-butanol as a solvent for freeze-drying: a case study.

1-(2-Chloroethyl)-3-sarcosinamide-1-nitrosourea, (SarCNU) (NSC-364432) is a new antitumor drug that is of interest to the National Cancer Institute. It is intended for use as an intravenous injection. Although SarCNU is sufficiently soluble in water to obtain the desired dosage, it is highly unstable. Its T(90) in aqueous solution at room temperature is less than 6 h. Neat tertiary butyl alcohol (TBA), a low toxicity, high vapor pressure and low melting solvent, was determined to be an excellent freeze-drying medium. Lyophilization of SarCNU from pure TBA produces a uniform cake composed of needle-shaped crystals. Thermal analysis and gas chromatography indicate that the cake contains less than 0.001% residual solvent. The SarCNU cake can be readily reconstituted with either water or an aqueous solution of 40% propylene glycol and 10% ethanol. The reconstituted solutions are stable for 4 and 13 h, respectively.

Synthesis and characterization of HPMA copolymer-aminopropylgeldanamycin conjugates.

Geldanamycin (GDM) is a benzoquinone ansamycin antibiotic with anticancer activity. The use of drug delivery systems based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing lysosomally degradable oligopeptide (GFLG) spacers results in an increased therapeutic efficacy of anticancer drugs. The objective of this study was to synthesize HPMA copolymer-GDM conjugates with anticancer activity and reduced toxic side-effect of the compound. 17-(3-Aminopropylamino)-17-demethoxygeldanamycin (AP-GDM) was synthesized and converted into a polymerizable GDM derivative, N-methacryloylglycylphenylalanylglycyl-17-(3-aminopropylamino)-17-demethoxygeldanamycin [MA-GFLG-(AP-GDM)]. The structures of AP-GDM and MA-GFLG-(AP-GDM) were validated by mass spectroscopy, elemental analysis, and two-dimensional nuclear magnetic resonance. MA-GFLG-(AP-GDM) was copolymerized with HPMA and N-methacryloyglycylglycine p-nitrophenylester by radical precipitation polymerization. Water-soluble HPMA copolymer-AP-GDM conjugates (M(r)=16 kDa) were obtained. Monoclonal antibody OV-TL16, which recognizes the OA-3 antigen expressed on the OVCAR-3 human ovarian carcinoma cell line, was optionally attached to the HPMA copolymer-AP-GDM conjugate. Cytotoxicity of polymer-bound AP-GDM (both targeted and non-targeted) was determined using OVCAR-3 and another human ovarian carcinoma cell line, A2780. The HPMA copolymer-AP-GDM conjugate was cytotoxic toward A2780 cells. Attachment of OV-TL16 antibody enhanced cytotoxicity of the conjugate toward OVCAR-3 cells.

Solubilization of NSC-639829.

Solubilization using pH combined with cosolvents, surfactants, and complexants are investigated for NSC-639829, an investigational anti-tumor agent. The intrinsic solubility of the drug is approximately 30 ng/ml and it has an ionizable dimethyl aniline group with an approximate base pK(a) of 5. Samples buffered at pH 1.0, 2.0, and 7.0 with various concentrations of the solubilizing agents were used to study the solubilization of NSC-629829 when present as charged and uncharged species. The solubilization of NSC-639829 was found to be much more effective when the drug was present primarily in ionized form. At pH values 1.0 and 2.0 where the surfactant (SLS) and complexant (SBEbetaCD) carried a negative charge enhanced solubilities of more than a million-fold were observed for the drug.

Solubilization of flavopiridol by pH control combined with cosolvents, surfactants, or complexants.

This study investigates the roles of both ionized and un-ionized species of flavopiridol in solubilization by complexation, micellization, and cosolvency. Control of pH was used in combination with surfactants (polysorbate 20 and polysorbate 80), cosolvents (ethanol and propylene glycol), as well as uncharged and anionic complexing agents [hydroxypropyl beta-cyclodextrin (HPbetaCD) and sulfobutyl ether beta-cyclodextrin (SBEbetaCD)] to solubilize flavopiridol. These combined techniques increase not only the solubility of the un-ionized flavopiridol but also the solubility of the ionized drug. This study confirms that previously developed equations effectively characterize the roles of pH, pK(a), and either complexation constant, micelle partition coefficient, or cosolvent solubilizing power in determining drug total aqueous solubility.

Solubilization of ionized and un-ionized flavopiridol by ethanol and polysorbate 20.

Because the ionized species is more polar than its un-ionized counterpart, it is often assumed that the ionized species of the drug does not make a meaningful contribution to solubilization by either cosolvents or surfactants. This report extends previous studies on solubilization of the ionic species by a combination of pH control and complexation to pH control and micellization and to pH control and cosolvency. The total aqueous solubility is expressed as the addition of the concentration of all contributing species: free un-ionized drug [Du], free ionized drug [Di], un-ionized drug micelle [DuM], and ionized drug micelle [DiM] for surfactant, and free un-ionized drug [Dcu] and free ionized drug [Dci] for cosolvent. The equations indicate that under certain conditions the ionized species can be more important in determining the drug total solubility than the un-ionized species. Flavopiridol, a weak base, is used to test these newly generated equations. As expected, the micellar partition coefficient and solubilization power for ionized flavopiridol are both less than those of the un-ionized species. However, at acidic pH, the solubilities of the ionized drug in surfactant micelles [DiM] and in cosolvent-water [Dci] are both much greater than that of the un-ionized drug. This difference is because the solubilization of the ionized drug is proportional to its aqueous solubility, and its solubility [Di] can be as much as 24-fold greater than that of the free un-ionized species [Du].

Spectrophotometric determination of acidity constants of 4-dedimethylamino sancycline (Col-3), a new antitumor drug.

A spectrophotometric technique was used to determine the acidity constants of 4-dedimethylamino sancycline (Col-3), a new antitumor drug. The apparent pKa values of Col-3 in 0.5% methanol aqueous media at approximately 25 degrees C with a constant ionic strength of 0.2 were calculated manually and graphically to be 5.64 +/- 0.17 (pKa1) and 8.35 +/- 0.07 (pKa2). In addition, the computer program SQUAD was used to confirm Col-3 pKa values. The pKa values obtained by SQUAD were pKa1 5.63 +/- 0.14 and pKa2 8.39 +/- 0.04. These results are in agreement with the tetracycline-like structure of Col-3.

Evaluation of intravenous flavopiridol formulations.

Flavopiridol is currently under consideration as a parenteral treatment of breast tumors. The solubility of this poorly water-soluble compound can be increased by a number of methods. However, because of its low water solubility, precipitation of the solubilized drug upon injection can be a problem. The potential of several flavopiridol formulations to precipitate is evaluated using a static serial dilution technique. It is shown that formulations containing up to 10 mg/mL of flavopiridol which produce negligible precipitation on dilution can be developed.

Activation of NF-kappaB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells.

PMN-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease. Epithelial cells respond to organisms such as Pseudomonas aeruginosa, the major pathogen in CF, by expressing the leukocyte chemokine IL-8. Experiments were performed using several different types of respiratory epithelial cells that demonstrate that ligation of ceramide-associated receptors on epithelial surfaces by P. aeruginosa pili is a major stimulus for the translocation of transcription factor nuclear factor (NF)-kappaB and initiation of IL-8 expression by epithelial cells. Using electrophoretic mobility shift assays and Western hybridizations, nuclear NF-kappaB was found shortly after epithelial cells were stimulated by either whole organisms, isolated pili, or antibody to the pilin receptor asialoGM1. IB3 cells, which express mutations in cystic fibrosis transmembrane conductance regulator (CFTR) (DeltaF508/W1282X), were noted to have significantly greater amounts of endogenous nuclear NF-kappaB, but not the transcription factor C/EBP, than CF cells corrected by episomal copies of normal CFTR (C-38) or IB3 cells grown at a permissive temperature (25 degreesC). Activation of NF-kappaB and subsequent IL-8 expression in epithelial cells can result from activation of at least two pathways: an exogenous signaling cascade that is activated by ligation of ceramide-associated adhesins such as P. aeruginosa pilin, or endogenous stimulation, suggested to be a consequence of cell stress caused by the accumulation of mutant CFTR in the endoplasmic reticulum.

Evaluation of in vitro precipitation methods.

Four in vitro precipitation methods were tested and evaluated using flavopiridol and diazepam formulations. The methods include static serial dilution, dynamic injection, and dropwise addition with and without stirring. The results generated from these methods are comparable and complementary. The static serial dilution method is most effective in quantifying the amount of precipitation and more descriptive of the formation and redissolution of the precipitate than the others. The dynamic injection method, however, has its merit in more realistically simulating the physiological environment of drug-blood interaction near the injection sites.

Combined effect of complexation and pH on solubilization.

Both pH control and complexation are widely used as solubilization techniques in drug formulation studies. Although these two techniques are often utilized in combination, few theoretical studies have shown why the combined approach would work better than either one alone. This study constructs a background in which both the pH effect and complexation constants are used to explain the synergism between these techniques. The total solubility is determined by the addition of the concentrations of the four components present in the solution: free un-ionized drug [Du], free ionized drug [Di], un-ionized drug complex [DuL], and ionized drug complex [DiL]. A detailed description of [Di] and [DiL] reveals that the complexation constants and the pH at which the drug may ionize are both critical. The weakly basic drug flavopiridol is used as a test compound to examine the validity of the equation. Although the complexation constant for ionized flavopiridol (Ki = 124 M(-1)) is less than one-third of that of the un-ionized species (Ku = 445 M(-1)), the solubility of the ionized drug complex [DiL] is 6-fold greater than that of the un-ionized drug complex [DuL]. This unexpected result is due to the 25-fold greater solubility of the ionized drug [Di] at pH 4.3 over that of the free un-ionized species [Du] at pH 8.4. The results of this and other complexation studies of several drugs taken from the literature lend the support to the following: If [Di]/[Du] >Ku/Ki, then [DiL] >[DuL].

Derivatization and high-performance liquid chromatographic analysis of pentaazapentacosane pentahydrochloride.

A rapid high-performance liquid chromatographic method for determination of the dansyl derivative of pentaazapen-tacosane (PAPC) pentahydrochloride has been developed. The chromatographic system uses a reversed-phase C8 column, a mobile phase of acetic acid buffer and acetonitrile and UV detection. The dansylation conditions were optimized with a pH of 11.0 and a 20-fold dansyl chloride excess. The yield of dansyl PAPC increased 10-fold as the reaction pH was changed from 9.5 to 10.5. Under derivatization conditions of pH 8.5-11.0 and 1-30-fold excess dansyl chloride only perdansyl PAPC was found.