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Despite the wide spread use of highly active antiretroviral therapy (HAART), mild forms of HIV-associated neuro cognitive disorders (HAND) remain commonplace. HAART treated patients now show low levels of viremia and more subtle yet biologically important signs of brain macrophage and microglial activation. Adjunctive therapeutic strategies are required to eliminate HIV-1 infection and suppress immune activation and its associated neuroinflammation. In this regard, cannabinoid receptor-2(CB2) activation is a promising means to attenuate HAND by inhibiting HIV replication, down regulating inflammation, and suppressing chemokine-like activity of viral neurotoxic proteins (for example, Tat and HIV-1gp120), and thereby prevent neuronal and synaptic loss. Inhibiting even low level HIV replication can attenuate neuronal injury by decreasing the production of neurotoxins. Down regulation of inflammation by CB2 activation is mediated through blunted activation of peri vascular macrophages and microglia; decreased production of tumor necrosis factor-α, chemokines and virotoxins. Down regulated neuroinflammation can decrease blood brain barrier permeability and leukocyte infiltration resulting in reduced neuronal injury. It is suggested that CB2 agonists may further attenuate HAND in HIVinfected patients on HAART. In addition, CB2 activation may also blunt brain injury by attenuating drug addiction.

Researchers have recently demonstrated the presence of anti-HIV-1 microRNAs (miR-28, miR-125b, miR-150, miR-223, and miR-382) in monocytes, macrophages, and CD4+ T cells, which are the primary targets of HIV infection. These miRNAs appear to regulate the level of infectivity of HIV-1 in the target cells, and thus have an impact on HIV-1 latency. The levels of these miRNAs are significantly higher in resting CD4+ T cells than those in active CD4+ T cells, whereas HIV-1 infectivity is greater in active than in resting CD4+ T cells. Similarly, the levels of these miRNAs are significantly higher in monocytes than in macrophages, whereas HIV-1 infectivity is greater in macrophages than in monocytes. Down-regulation or inhibition of the activity of these miRNAs can promote replication of latent HIV-1 in resting CD4+ T cells and in monocytes. Recently, morphine was shown to down regulate the expression of anti-HIV miRNAs (miRNA-28, 125b, 150, and 382) in cultured human monocytes and this effect of morphine was mediated via activation of mu opioid receptors (MOR). In addition, levels of these anti-HIV miRNAs were significantly lower in the peripheral blood mononuclear cells (PBMCs) isolated from heroin-dependent subjects than those from control subjects. These findings raise an important question: Does morphine have potential to activate latent HIV-1 in resting CD4+ T cells and macrophages, including microglia of human subjects maintained on highly active antiretroviral therapy (HAART)? Further research is required to answer this question.

In the pre-highly active antiretroviral therapy (HAART) era, prenatal "vertical" mother-to-child transmission (MTCT) of HIV was about 25% and exposure of pregnant mothers to drugs of abuse (illicit drugs and tobacco smoking) was a significant contributory factor of MTCT. However, with the introduction of HAART, the rate of MTCT of HIV has decreased to less that 2%. But, it is estimated that currently about 5.1% of pregnant women use illicit drugs and 16.4% smoke tobacco. The residual prevalence of MTCT is of concern and may be related to this continued prevalence of substance use among pregnant mothers. In this report, we review and present evidence that supports the hypothesis that drugs of abuse do have the potential to increase MTCT of HIV in the presence of HAART. Exposure to drugs of abuse during pregnancy may increase MTCT of HIV through a variety of mechanisms that are addressed in detail including possible damage to the placenta, induction of preterm birth, and increasing maternal plasma viral load though a variety of putative mechanisms such as: (a) promoting HIV replication in monocyte/macrophages; (b) increasing the expression of CCR5 receptors; (c) decreasing the expression of CCR5 receptor ligands; (d) increasing the expression of CXCR4 receptors; (e) increasing the expression of DC-SIGN; (f) impairing the efficacy of HAART through drug-drug interaction; and (g) promoting HIV mutation and replication through non-adherence to HAART.

We were gratified by the interest expressed in publishing a large number of presentations from the NIDA organized Workshop on "Natureceuticals (Natural Products), Nutraceuticals, Herbal Botanicals, Psychoactives: Drug Discovery and Drug-Drug Interactions". The number of manuscripts received necessitated two volumes of proceedings. In this brief summary of the second volume, we present an introduction to the roles of organizations such as National Center for Complementary and Alternate Medicine and Office of Dietary Supplements, both at the National Institutes of Health, and the Food and Drug Administration. These agencies are involved in research and regulation of dietary supplements and related products. Next, a brief summary of each of the fifteen articles is provided. The first four articles are related to regulatory and standardization aspects: issues related to botanicals (Khan); USP and dietary supplements (Srinivasan); dietary supplement reference materials (Sander et al.); and proposed cGMPs and the scientific basis behind the proposed regulations by FDA (Melethil). The next three articles relate to the methodologies employed in research: LC/MS for the pharmacokinetic analysis polyphenols from dietary supplements (Barnes et al.); proteomic analysis of grape seed extract (Kim et al.); and a nematode model, C. elegans, in Alzheimer's and ginkgo biloba extract for mechanistic studies; another model, a hepatocyte tissue culture model for drug herbal interaction, is reviewed later and presented by Venkataramanan. The next four chapters are on specific dietary supplements: green tea and the polyphenolic catechins (Zaveri); curcumin (Maheswari et al.); tocotrienols (alpha-tocotrienol, Sen and Roy), gamma-tocotrienol (Sree Kumar et al.). This topic is followed by drug interaction studies: in vitro and in vivo assessment methodologies (Venkataramanan); flavonoid-drug interactions (Morris); MDR and CYP3A4-mediated drug-herb interaction (Pal and Mitra); and evidence-based examination of drug-herb interaction (Chavez and Chavez).

Contemporaneously with genomics and proteomics technologies, lipidomics may be recognized as the next important emerging technology. The emergence of this important area of "omics" could very well mark the beginning of "the decade of the lipids." A workshop on lipidomics was held in Washington, DC, by the National Institute on Drug Abuse (NIDA) in April 2004. The goal of the workshop was to bring together scientists working at the frontier of lipid research, to discuss their findings in this area and to promote "lipidomics," in general, but also with a special focus on its application to drug abuse research and development of therapies to treat addiction.

The use of illicit and licit drugs during pregnancy is a major public health concern because of potential adverse effects on the fetus and the risk to maternal health. Because the placenta is the primary link between the mother and the conceptus and is essential for the growth and survival of the fetus, abnormalities in placental formation and function resulting from drug use could have a major influence on pregnancy outcome. At present, little information is available on the impact of abused drugs on placental biology alone or in combination with other "host" factors (eg, stress, infections). This prompted the National Institute on Drug Abuse (NIDA) to convene a meeting of experts in placental biology to review cutting-edge research with the mission to translate existing information to new clinical and research initiatives in the drug abuse field. This report summarizes the presentations and research recommendations resulting from the workshop discussions.

Although much effort has been devoted to opioid research since the identification of enkephalins, understanding of the physiological importance and mechanisms of action of endogenous opioids lags behind understanding of opiate alkaloids such as morphine. In recent years, several novel approaches have been refined with promise for the successful development of the long-awaited nonaddicting analgesics that act at the opioid delta receptor. The present communication reviews these efforts.

Cannabinoids have been known to exhibit a wide variety of biological effects. Over the past fifty years numerous analogs were synthesized in an attempt to understand the structural requirements for each cannabinoid activity. Only recently, however, some important findings have focused new attention on this field of research. These findings include: (a) The development of novel "non-classical" potent cannabinoid analogs which exhibit similar pharmacological profiles with their "classical" counterparts; (b) The demonstration that there are specific cannabinoid binding sites in cell cultures as well as in mammalian brains; (c) Biophysical studies related to the interactions of cannabinoids with membranes which lead to a better understanding of those molecular properties which are required for cannabinoid activity; (d) Detailed and uniform pharmacological testing on a sizeable number of analogs allowing for a more detailed dissection of the cannabinoid effects and respective "structure activity relationships." The newly increased interest in cannabinoid research opens the door for a better understanding and potential treatment in cases of abuse as well as novel therapeutic opportunities through the design and synthesis of pharmacologically more selective analogs.

Synthesis of two copolymers, H-(phi-Pro-Gly-Gly)n-Val-OMe and H-(phi'-Pro-Gly-Gly)n-Val-OMe, where phi is Val or Lys and phi' is Val or Glu is described. Cross-linking between the two copolymers is achieved by a coupling reaction between the epsilon-amino groups of the lysine containing copolymer and delta-carboxyls of the glutamic acid of the other copolymer. The cross-linking reaction was performed during a temperature elicited phase separation with flow orientation of the copolymers. Coacervation of the intermediate polymers is presented, as is the scanning electron micrograph of the insoluble cross-linked product and its calcifiability as determined with electron probe microanalysis. The purity of the key intermediates and polymers is demonstrated by the usual analytical methods. Carbon-13 magnetic resonance spectra of the intermediate monomer units are included to validate the purity of their synthesis.

Synthetic repeat polypeptides analogous in sequence to the valine-rich regions of elastin have been tested as substrates for purified bovine aorta lysyl oxidase. These polypeptides, HCO(phi-Pro-Gly-Gly)n-Val-OMe, HCO(Val-Pro-Gly-phi-Gly)n-Val-OMe, and HCO-Val-(Ala-Pro-Gly-phi-Gly-Val)n-OMe, where phi = Val or Lys at approximately a 4:1 ratio and where n greater than or equal to 40, are models of the tetra-, penta-, or hexapeptide repeat sequences found in elastin. alpha-Aminoadipic delta-semialdehyde is generated in each of these upon incubation with lysyl oxidase at 37 degrees C, whereas the aldol and anhydrolysinonorleucine bifunctional cross-linkages were formed only in the incubation of enzyme with polypentapeptide. Incubation of the polypentapeptide at 55 degrees C, which enhances coacervation of the peptide, increases aldehyde formation and generates a much higher ratio of cross-linkages to aldehyde than occurred at 37 degrees C. These results demonstrate that lysyl oxidase can oxidize lysine in synthetic polypeptides and suggest important conformational aspects of lysyl oxidase substrates which may control substrate potential as well as the ability of peptidyl aldehyde, once formed by the enzyme, to condense to cross-linkage products.

The FT-IR (Fourier Transform Infrared) Spectrum of [Met 5]-enkephalinamide in aqueous solution shows the presence of both the beta-turn and beta-sheet conformations. The beta-turn and beta-sheet conformations of enkephalins have been proposed to play a role in receptor selectivity. Addition of ethanol alters these secondary structural features and hence the effect of ethanol on ligand-receptor interaction may be mediated primarily through conformational changes of the ligand rather than those of the receptor.

Syntheses of two sequential polytetrapeptide models, H-(Val-Ala-Pro-Gly)n-Val-OMe and H-(Val-Pro-Gly-Gly)n-Val-OMe via the p-nitrophenyl ester method are described. The p-nitrophenyl ester method gave high yields (85%--100%) of large molecular weight polymers. H-(Val-Pro-Gly-Gly)n-Val-OMe exhibited the interesting property of coacervation on raising the temperature of aqueous solutions while H-(Val-Ala-Pro-Gly)n-Val-OMe precipitates irreversibly under similar conditions. Whereas non-dialyzed lower molecular weight polymers of H-(Val-Pro-Gly-Gly)n-Val-OMe with n = 8 to 40 did not coacervate, but did show a transition to increased intramolecular order on raising the temperature of aqueous solutions above 50 degrees C, the dialyzed higher molecular weight polymer, n greater than 40, does coacervate beginning at about 50 degrees C. This demonstrates the molecular weight dependence of coacervation and also suggests the importance to coacervation of side chain interactions in the Val-Pro sequence. The increase in intramolecular order, seen as the formation of a 14-atom hydrogen-bonded ring, occurs simultaneously with the Val-Pro hydrophobic side chain association.

The synthesis of several sequential oligomer models of collagen is reported. Polytripeptides H(-Pro-Gly-Leu)n OH, H(-Pro-Gly-Phe)n OH, H(-Pro-Sar-Gly)n OH, H-(Sar-Pro-Gly-)n -OH, H(-Pro-Gly-V-AL-)N OH and H(-Gly-Val-Pro)n -oh were synthesized by the active ester method. Preliminary fractionation and mol.wt. determination showed typical gaussian distributions. Good yields were obtained in the mol.wt. range of 4,000-6,000, except in the case of H(-Pro-Sar-Gly-)n OH which yielded molecular weights over 20,000.

Levothyroxine sodium was estimated from tablet formulations of levothyroxine sodium and liotrix (liothyronine sodium-levothyroxine sodium combination tablet). The procedures consisted of the addition of 3,3-,5-triiodothyronine as the internal standard to the pulverized sample, followed by an acidic butanol extraction, evaporation, and injection onto a muBondapak reversed-phase high-performance liquid chromatographic column. The eluent was methanol-water-phosphoric acid (50:50:0.1), and the effluent was monitored by UV detection at 254 nm. A standard linear calibration curve was obtained for direct standard solutions equivalent to 18-225 micrograms of levothyroxine sodium/tablet. The procedure is sensitive enough for single-tablet analysis. Using this procedure, content uniformity studies were performed on liothyronine sodium tablets, levothyroxine sodium tablets, and liotrix tablets. The procedure also was adapted for conducting dissolution studies on levothyroxine sodium tablets in deionized water using the rotating-paddle method.

In order to evaluate the in vitro dissolution and in vivo bioavailability relationship for furosemide, a bioequivalence study was carried out. Furosemide (40 mg) was administered orally to 12 normal volunteers in a 6 x 6 crossover design using six products (five tablets and one solution) obtained from three pharmaceutical companies. Plasma and urine concentrations of furosemide were quantitated by high-performance liquid chromatography (HPLC). Plasma furosemide profiles were analyzed by non-compartmental methods. Compared to the oral solution, all of the formulations exhibited lower peak furosemide concentrations, longer mean residence times, and, in some cases, diminished bioavailability (range, 66-96%). Similar results were obtained when the reference product (a rapidly dissolving tablet) was used as the standard. All of the products failed the 75/75 rule when compared to either reference standard, apparently because of large intersubject variability. The total amount of furosemide excreted in urine could be associated with the percentage drug dissolved (in vitro) at 30 min. The pH 5.6 dissolution medium (compared to pH 4.6) appears to be an appropriate test medium for assuring batch uniformity and bioequivalence of furosemide products.

Modifications of existing rapid high-performance liquid chromatographic procedures for the determination of furosemide in plasma were made in order to achieve greater sensitivity. To a small volume of plasma was added in internal standard structurally related to furosemide. Then, following previously described procedures, acetonitrile was added to precipitate the proteins and the clear supernatant was separated. However prior to injection of the supernatant the pH and composition of the sample were adjusted. This modification of the sample enabled an injection volume of up to 300 microliters of the supernatant to be injected onto the chromatographic column. The effluent was monitored spectrofluorimetrically. A standard linear calibration curve with a mean precision of +/- 4.4% was obtained for plasma samples containing 20--900 ng/ml of furosemide. Two structurally related compounds were used as internal standards in the furosemide assay.

Syntheses of two sequential polypeptides H-(Ala-Pro-Gly-Gly)n-Val-OMe and H-(Ala-Pro-Gly-Val-Gly)n-Val-OMe via the p-nitrophenyl active ester procedure are reported. The two polymers were obtained in good yields and the polymers were shown to be of large molecular weights, n greater than 40. These two polypeptides were synthesized as analogs of the two coacervating sequential polypeptides H-(Val-Pro-Gly-Gly)n-Val-OMe, and H-(Val-Pro-Gly-Val-Gly)n-Val-OMe, in which the Val-l residue is replaced by an Ala-l residue. H-(Ala-Pro-Gly-Gly)n-Val-OMe did not coacervate even at as high a temperature as 100 degrees, and H-(Ala-Pro-Gly-Val-Gly)n-Val-OMe did not coacervate; however, it precipitated irreversibly around 65--70 degrees C. This suggests the critical role of the Val-Pro hydrophobic side chain interaction in coacervation.

The synthesis of two collagen models (Pro-Pro-Ala)n and (Pro-Pro-Val)n is reported. Preliminary examination suggests that (Pro-Pro-Ala)n may exhibit some properties of collagen-like polypeptides whereas (Pro-Pro-Val)n, does not have a collagen-like character.

FT-IR spectroscopic studies of dynorphin A(1-13) in H2O and D2O are utilized to derive the aqueous phase secondary structure of the opioid peptide. Resolution enhancement of the amide I region of dynorphin A(1-13) in H2O revealed a doublet at 1652 cm-1 and 1669 cm-1 which are interpreted as indicative of "unordered" and extended structures. From FT-IR and 1H NMR deuterium exchange studies, the peptide NH groups appeared to be solvent accessible which is suggestive of an essentially extended structure with aperiodically interwoven "unordered" structure. The results are consistent with Raman Spectroscopic (Rapaka et al., (1987) Int. J. Peptide Protein Res. 30:284-287) and 2D NMR studies (Huang et al. submitted), from our laboratory.

Hydralazine (1-hydrazinophthalazine) produces skeletal defects resembling those observed in experimentally induced manganese deficiencies. Since glycosylation of collagen, a step preceding its secretion, requires Mn2+, the effect of hydralazine on collagen secretion and the formation hydroxylysine glycosides was examined in explants of embryonic chicken long-bone rudiments. Auto-radiographic studies showed that hydralazine blocked collagen secretion. Secretion was restored by Fe2+ alone or Fe2+ + Mn2+ but not by Mn2+ alone, suggesting that a Fe2+-requiring step was involved. Biochemical analyses showed that hydralazine inhibited the formation of hydroxylysine, a step requiring Fe2+, but it did not inhibit the formation of hydroxylysine-glocosides by Mn2+-requiring steps, although the reaction was inhibited in vitro. Hydralazine also failed to inhibit intracellular mucopolysaccharide synthesis which involves several Mn2+-requiring enzymes. These observations suggest that the deleterious effects of hydralazine on bones are caused by its inhibition of hydroxylation steps in collagen synthesis.

Palladium ions, administered as PdSO4, markedly affect the incorporation of L-[3,4-3H2] proline into non-dialyzable fractions in 10-day chick embryo cartilage explants with a 55-65% reduction in the concentration range 0.06-0.6 mM. Under these conditions the synthesis of [3H]hydroxyproline was nearly completely inhibited. Experiments with prolyl hydroxylase (EC 1.14.11.2) indicated a strong irreversible inhibition of the enzyme with a competition between Fe2+ and Pd2+. The Ki for the inhibition was 0.02 mM. Pd2+-treated enzyme remained inactive after extensive dialysis. These studies suggest that Pd2+ may inhibit collagen synthesis by replacing Fe2+ in the active site of prolyl hydroxylase and forming strong complexes with the enzyme. These studies also point to a potential mechanism of Pd2+ toxicity.

The conformation of a hexapeptide sequence occurring in tropoelastin is discussed from the results obtained using a combined approach of theoretical conformational energy calculations on HCO-Val-Ala-Prb-Gly-OMe and 1h nmr studies on t-Boc-Val-Ala-Pro-Gly-Val-Gly-OMe in a dilute solution of methanol. Both studies have reasonable concurrence with respect to the preferred conformation of the hexapeptide and an analysis of the combined results suggests that the hexapeptide is stabilized by a beta-turn involving the Ala1,iC=O and Val4,iNH groups and a gamma-turn involving Gly5,iC=O and Gly3,iNH groups. A weaker interaction between Gly3,iC=O and Gly5,iNH groups is also found to be possible. Conformational features of the first valyl residue in the sequence Val-Ala-Pro-Gly-Val-Gly and the last valyl residue in Ala-Pro-Gly-Val-Gly-Val are compared and found to have similar torsion angles. The implications of such a similarity are discussed with respect to the conformation of the polyhexapeptide.

Conformational features of dynorphin A-(1-13) were examined by laser Raman spectroscopy. Dynorphin A-(1-13) appears to have a mixture of extended beta-pleated sheet and "random" structure.