Pharmaceutical Research

Purpose. The mobility of protein in powders at different hydration levels was studied in relation to aggregation and activity. Methods. Magic angle spinning 13C, 15N, 1H, 2H, and 17O NMR techniques were used to determine changes in the mobility of surface residues in proteins as a function of hydration and related to changes in activity. NMR relaxation measurements of high frequency (ω0, T1) and low frequency (ω1, T1p) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced aggregation and enzymatic activity of the lyophilized proteins was determined by high performance size exclusion chromatography and bioassays. Results. There was little change in T1p observed with increasing humidity. The results show, however, that there is a decrease in T1, for DNase, insulin and lysozyme at relative humidities ranging from 0−98%, and we propose that the reduction in T1, is related to the aggregation susceptibility of proteins during storage at different humidities. The water mobility was determined directly using 17O NMR experiments. We found that as the amount of weakly-bound water increases, the protein surface mobility decreases and is coupled with increased aggregation. Aggregation measurements at different humidities were correlated with bioassays for lysozyme and found to be consistent with the hydration data. Conclusions. Mobility of protein molecules was determined by solid-state NMR over a wide range of % RH and it was found that water content leads to a change in mobility of protein molecules. The aggregation and activity of proteins were strongly correlated to change in molecular mobility.

Purpose. To evaluate the magnitude of the solubility advantage foramorphous pharmaceutical materials when compared to their crystallinecounterparts.Methods. The thermal properties of several drugs in their amorphousand crystalline states were determined using differential scanningcalorimetry. From these properties the solubility advantage for theamorphous form was predicted as a function of temperature using a simplethermodynamic analysis. These predictions were compared to theresults of experimental measurements of the aqueous solubilities of theamorphous and crystalline forms of the drugs at several temperatures.Results. By treating each amorphous drug as either an equilibriumsupercooled liquid or a pseudo-equilibrium glass, the solubilityadvantage compared to the most stable crystalline form was predicted to bebetween 10 and 1600 fold. The measured solubility advantage wasusually considerably less than this, and for one compound studied indetail its temperature dependence was also less than predicted. It wascalculated that even for partially amorphous materials the apparentsolubility enhancement (theoretical or measured) is likely to influencein-vitro and in-vivo dissolution behavior.Conclusions. Amorphous pharmaceuticals are markedly more solublethan their crystalline counterparts, however, their experimental solubility advantage is typically less than that predicted from simplethermodynamic considerations. This appears to be the result of difficulties indetermining the solubility of amorphous materials under trueequilibrium conditions. Simple thermodynamic predictions can provide a useful indication of the theoretical maximum solubility advantage foramorphous pharmaceuticals, which directly reflects the driving forcefor their initial dissolution.

Purpose. The efficiency of encapsulation of water-soluble drugs in biodegradable polymer is often low and occasionally these microcapsules are associated with high burst effect. The primary objective of this study is to develop a novel microencapsulation technique with high efficiency of encapsulation and low burst effect. Method. Pentamidine was used as a model drug in this study. Pentamidine/polyvinyl alcohol (PVA) hydrogel was prepared by freeze-thaw technique. Pentamidine loaded hydrogel was later microencapsulated in poly(lactide-co-glycolide) (PLGA) using solvent evaporation technique. The microcapsules were evaluated for the efficiency of encapsulation, particle size, surface morphology, thermal characteristic, and drug release. Results. Scanning Electron Microscope (SEM) studies revealed that the microcapsules were porous. The microcapsules were uniform in size and shape with the median size of the microcapsules ranging between 27 and 94 μm. The samples containing 10% PLGA showed nearly three times increase in drug loading (18-53%) by increasing the hydrogel content from 0-6%. The overall drug release from the microencapsulated hydrogel, containing 3% and 6% PVA, respectively, was significantly lower than the control batches. Conclusions. The use of a crosslinked hydrogel such as PVA can significantly increase the drug loading of highly water-soluble drugs. In addition, incorporation of the PVA hydrogel significantly reduced the burst effect and overall dissolution of pentamidine.

Purpose. The aims of this study were (i) to elucidate the transport mechanisms involved in drug release from hydrophilic matrices; and (ii) to develop an improved mathematical model allowing quantitative predictions of the resulting release kinetics. Methods. Our previously presented model has been substantially modified, by adding: (i) inhomogeneous swelling; (ii) poorly water-soluble drugs; and (iii) high initial drug loadings. The validity of the improved model has been tested experimentally using hydroxypropyl methylcellulose (HPMC)-matrices, containing either a poorly or a freely water-soluble drug (theophylline or chlorpheniramine maleate) at various initial loadings in phosphate buffer pH 7.4 and 0.1 N HCl, respectively. Results. By overcoming the assumption of homogeneous swelling we show that the agreement between theory and experiment could be significantly improved. Among others, the model could describe quantitatively even the very complex effect on the resulting relative release rates (first slowing down, then accelerating drug release) observed when increasing the initial loading of poorly water-soluble drugs. Conclusions. The practical benefit of this work is an improved design model that can be used to predict accurately the required composition and dimensions of drug-loaded hydrophilic matrices in order to achieve desired release profiles, thus facilitating the development of new pharmaceutical products.

Acute doxorubicin-loaded nanoparticle (DXNP) renal toxicity was explored in both normal rats and rats with experimental glomerulo-nephritis. In normal rats, 2/6 rats given free doxorubicin (DX) (5 mg / kg) died within one week, whereas all control animals and all rats having received free NP or DXNP survived. A 3 times higher proteinuria appeared in animals treated with DXNP than in those treated with DX. Free NP did not provoke any proteinuria. Two hr post-injection, DXNP was 2.7 times more concentrated in kidneys than free DX (p<0.025). In rats with immune experimental glomerulonephritis, 5/6 rats given DX died within 7 days, in contrast to animals treated by DXNP, NP, or untreated, which all survived. Proteinuria appeared in all series, but was 2-5 times more intense (p>0.001) and prolonged after doxorubicin treatment (400-700 mg / day), without significant difference between DXNP and DX. Rats treated by unloaded NP behaved as controls. These results demonstrate that, in these experimental conditions, DXNP killed less animals than free DX, despite of an enhanced renal toxicity of the former. Both effects (better survival and nephrosis) are most probably related to an enhanced capture of DXNP by cells of the mono-nuclear phagocyte system, including mesangial cells.

Purpose. In the summer of 1998, Norvir semi-solid capsules supplies were threatened as a result of a new much less soluble crystal form of ritonavir. This report provides characterization of the two polymorphs and the structures and hydrogen bonding network for each form. Methods. Ritonavir polymorphism was investigated using solid state spectroscopy and microscopy techniques including solid state NMR, Near Infrared Spectroscopy, powder X-ray Diffraction and Single crystal X-ray. A sensitive seed detection test was developed. Results. Ritonavir polymorphs were thoroughly characterized and the structures determined. An unusual conformation was found for form II that results in a strong hydrogen bonding network A possible mechanism for heterogeneous nucleation of form II was investigated. Conclusions. Ritonavir was found to exhibit conformational polymorphism with two unique crystal lattices having significantly different solubility properties. Although the polymorph (form II) corresponding to the “cis” conformation is a more stable packing arrangement, nucleation, even in the presence of form II seeds, is energetically unfavored except in highly supersaturated solutions. The coincidence of a highly supersaturated solution and a probable heterogeneous nucleation by a degradation product resulted in the sudden appearance of the more stable form II polymorph.

The physical characteristics of polymerized liposomes for potential use as an oral drug delivery system were examined in vitro. The trap efficiency in monomeric liposomes composed of 1,2-di (2,4-octadecadienoyl) phosphatidylcholine was increased from 3% for original multilamellar vesicles to 35% for freeze-thaw treated liposomes. Polymerized liposomes with azobis (isobutyronitrile) and azobis (2-amidinopropane) hydrochloride as radical initiators showed complete stability against solubilization by Triton X-100, a detergent chosen to mimic bile salts. Release rates of 14C-BSA and 14C-sucrose in media simulating the gastro-intestinal fluids was 50% less than from regular liposomes composed of hydrogenated egg phosphatidylcholine mixed with cholesterol (molar ratio 1:1), which can be regarded as one of the most stable types of regular liposomes. It was estimated that, when administered orally, polymerized liposomes can reach the intestine while maintaining their vesicle structure and keeping at least 75% of their original content.

Oxidation is one of the most common degradation pathways for active pharmaceutical ingredients (APIs) in pharmaceutical formulations, mostly involving 1-electron processes via peroxy radicals and 2-electron processes by peroxides. In liquid pharmaceutical formulations, several factors can impact oxidative instabilities including pH, excipient impurities, headspace oxygen, and the potential for photo-oxidation. Photo-oxidation can be particularly challenging to characterize given the number of oxidative mechanisms which can occur. This was observed during formulation development of a new chemical entity, MK-1454, where a degradation peak was observed during photostability studies which was not previously observed during peroxide and peroxyradical forced stress studies. To gain a fundamental understanding of reactive oxygen species generation and its role in degradation of MK-1454, experiments were performed with materials which either generate or measure reactive oxygen species including organic hydroperoxides, singlet oxygen, and superoxide to fundamentally understand a photodegradation mechanism which was observed in the original formulation. LC-MS experiments further elucidated the structure and mechanism of this observed degradation pathway. A clear relationship between the decrease in dissolved oxygen after light exposure and the loss of MK-1454 was established. The data indicate that singlet oxygen is the most likely contributor of a particular photodegradation product. The singlet oxygen was generated by the inactive ingredients in the formulation, and LC-MS confirm this as the most likely pathway. This work highlights the importance of understanding photochemical degradation of APIs in solution formulations and provides approaches which can better elucidate those mechanisms and thereby control strategies.

The β-adrenergic receptors (βAR) play important roles in cardiovascular function and disease, and both agonists and antagonists are widely used in various settings for treatment of cardiovascular disease. Both the β1AR and β2AR genes have several polymorphisms that are common in the population and result in encoding of different amino acids. More importantly, in vitro functional studies suggest that these polymorphisms have functional significance. In this review we summarize the literature on the relationship between the βAR polymorphisms and cardiovascular disease as well as the literature on the impact of these polymorphisms on drug response. Additionally, the polymorphisms in both the β1AR and β2AR genes are in linkage disequilibrium; thus, the relevance of single polymorphism vs. haplotype analysis is discussed. Further study of the βAR genetic polymorphisms is likely to enhance our understanding of cardiovascular disease and improve our use of β-agonists and β-antagonists in treatment of cardiovascular disease.

Purpose. To define the tubular localization and tissue distribution of PEPT1 (low-affinity, high-capacity transporter) and PEPT2 (high-affinity, low-capacity transporter) in rat kidney. Methods. mRNA expression of PEPT1 and PEPT2 was assessed with reverse transcription-polymerase chain reaction (RT-PCR) methods using cDNA prepared from microdissected nephron segments in rat. Tissue localization of rat renal PEPT1 and PEPT2 mRNA was further assessed by in situ hybridization with radiolabeled probes. Results. RT-PCR analysis of microdissected segments from rat nephron showed that both PEPT1 and PEPT2 are confined to the proximal tubule. While PEPT1 is specific for early regions of the proximal tubule (pars convoluta), PEPT2 is overwhelmingly but not exclusively expressed in latter regions of the proximal tubule (pars recta). All other segments along the nephron were negative for PEPT1 or PEPT2 mRNA transcripts. These findings were supported by in situ hybridization results in which PEPT1 was selectively expressed in kidney cortex and PEPT2 in the outer stripe of outer medulla. Conclusions. Contrary to current opinion, the data suggest that peptides are handled in a sequential manner in proximal regions of the nephron, first by the low-affinity, high-capacity transport system and second by the high-affinity, low-capacity transport system.