Enhancer Potency Research Articles

Silicone Elastomer Uptake Method for Determination of Free 1‐Alkyl‐2‐Pyrrolidone Concentration in Micelle and Hydroxypropyl‐β‐Cyclodextrin Systems Used in Skin Transport Studies

Previous investigations in our laboratory demonstrated how the polar head group and alkyl chain of amphiphilic chemical skin permeation enhancers contribute to enhancer potency. In those studies enhancers with n-alkyl chain lengths of eight or less were investigated. In order to investigate enhancers with longer n-alkyl chain lengths, enhancer-solubilizing agents should be considered. Corticosterone (CS) flux enhancement along the lipoidal pathway of hairless mouse skin (HMS) was determined with the enhancers 1-hexyl- (HP), 1-octyl- (OP), 1-decyl- (DP), and 1-dodecyl-2-pyrrolidone (DoP) solubilized in 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol-2000] (DSPE) micelles or in hydroxypropyl-beta-cyclodextrin (HPbetaCD). The free CS, HP, OP, DP, and DoP aqueous concentrations in the DSPE micelle and HPbetaCD systems were determined using a partitioning method. Comparisons of the enhancer potencies based on the free concentration of the enhancers revealed a nearly semi-logarithmic linear relationship between enhancer potency and the carbon number of the alkyl chain length with a slope of approximately 0.55. The observed n-alkyl chain length dependency in the aqueous phase is consistent with the hydrophobic effect. This study shows that longer chain enhancers may be studied by employing a solubilizing system, and free enhancer concentration in these systems can be determined with the aid of the silicone elastomer uptake method.

Mechanistic Studies of Branched-Chain Alkanols as Skin Permeation Enhancers

As part of a long-term effort to understand the structure/function relationship between chemical permeation enhancers and skin permeation enhancement, the present study examined the influence of hydrocarbon chain branching on the effectiveness of skin permeation enhancers of the type that possesses a polar group (e.g., the hydroxyl group) attached to a hydrocarbon chain(s). The effects of x-hexanol, x-heptanol, x-octanol, and x-nonanol (where x is the position of the hydroxyl group ranging from 1 up to 5) on the transport of a probe permeant, corticosterone, across hairless mouse skin (HMS) were investigated. Isoenhancement concentrations are defined as the aqueous concentrations for which different enhancers induce the same extent of permeant transport enhancement, E, across the lipoidal pathway of stratum corneum (SC). The isoenhancement concentrations of 2-alkanol, 3-alkanol, 4-alkanol, and 5-alkanol to induce E=10 were approximately 1.9-, 2.6-, 3.1-, and 3.9-fold higher, respectively, than those of the 1-alkanols of the same molecular formula. This suggested that the branched-chain alkanols have lower enhancer potency than the 1-alkanols of the same molecular formula; the potency decreases as the hydroxyl group moves from the end of the chain towards the center of the enhancer alkyl chain. To further investigate the mechanism(s) of action of the branched-chain alkanols as skin permeation enhancers, the equilibrium uptake of the enhancers into the hairless mouse skin stratum corneum (HMS SC) from aqueous enhancer solutions of E=10 was determined. The data from these experiments provided a direct measure of the “intrinsic” potency of the enhancer. In the same experiments, the equilibrium partitioning (distribution) of a surrogate permeant, estradiol (E2β), into the HMS SC was also determined and compared to the partitioning from PBS (no enhancer present). The uptake amounts (micromole/mg SC) for 1-alkanols into the intercellular lipids of the SC were found to be essentially the same at their isoenhancement concentrations. However, at their isoenhancement concentrations, the uptake amounts of the branched-chain alkanols into the intercellular lipids of HMS SC were higher than those of the 1-alkanols. These results support the view that: (1) the intrinsic potencies of the 1-alkanols are essentially the same and independent of their 1-alkyl chain length at their isoenhancement concentrations, (2) the intrinsic potencies of the branched-chain alkanols are lower than those of the normal alkanols, and (3) branching of the alkyl chain reduces the ability of the enhancer to effect lipid fluidization in the SC lipid lamellae at the target site(s). The enhancement effects of the branched-chain alkanols and the 1-alkanols at their isoenhancement concentrations upon E2β partitioning into the SC intercellular lipids were found to be approximately the same and in the range of five- to eight-fold enhancement. The constancy of this enhancement for E2β partitioning suggests that the mechanism of enhancement action for the branched-chain alkanols and the 1-alkanols are the same. Additionally, a good correlation of the intercellular lipid/PBS partition coefficients of both the branched-chain alkanols and the 1-alkanols with the n-octanol/PBS partition coefficients was found. This supports the view that the chemical microenvironment of the polar head group and the alkyl group of the studied enhancers at the site of skin permeation enhancer action in the SC lipid lamellae can be represented by water-saturated n-octanol for both the branched-chain alkanols and the 1-alkanols. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association

Exosomes as a tumor vaccine: enhancing potency through direct loading of antigenic peptides.

Exosomes secreted by dendritic cells (DCs) contain MHC-I, MHC-II, and other accessory molecules required for antigen presentation to T cells. Previous studies have shown that exosome MHC-I "indirectly" loaded by adding peptides to DC cultures are immunogenic. However, analysis of peptide binding was not performed to link T-cell-stimulating activity with the amount of MHC-I/peptide complexes on the exosomes. In this study, we measured peptide binding to MHC-I under different loading conditions and tested the exosomes' potencies in T-cell activation assays. We demonstrate that MHC-I on purified exosomes can be directly loaded with peptide at much greater levels than indirect loading. The direct loading method performed in mildly acidic conditions was effective even in the absence of exogenous beta2m. This increase in peptide binding greatly enhanced exosome potency, allowing us to further study the biologic activity of exosomes in vitro. In the presence of antigen-presenting cells (APC), exosomes directly loaded with the HLA-A2 restricted MART1 tumor peptide stimulated an HLA-A2/MART1 specific T-cell line. The T cells responded to exosomes using HLA-A2neg APC, demonstrating transfer of functional MHC-I/peptide complexes and not peptide alone to APC. MHC-II molecules, which are abundantly expressed on DC exosomes, were also functionally loaded under the same conditions as MHC-I. This feature allows for delivery of multiple peptide antigens that can stimulate both CD8+ cytotoxic T cells as well CD4+ T helper cells critical for an effective antitumor response. The optimized loading conditions and the ability to transfer both MHC-I and MHC-II antigens to APC have led to the development of exosomes as an "acellular" immunotherapy approach currently being tested in clinical trials.

Structure–Activity Relationship for Chemical Skin Permeation Enhancers: Probing the Chemical Microenvironment of the Site of Action

Studies were previously conducted in our laboratory on the influence of n‐alkanols, 1‐alkyl‐2‐pyrrolidones, N,N‐dimethlyalkanamides, and 1,2‐alkanediols as skin permeation enhancers on the transport of a model permeant, corticosterone (CS). The experiments were conducted with hairless mouse skin (HMS) in a side‐by‐side, two‐chamber diffusion cell, with enhancer present in an aqueous buffer in both chambers. The purpose of the present study was to extend these studies and investigate in greater detail the hypothesis that a suitable semipolar organic phase may mimic the microenvironment of the site of enhancer action, and that the enhancer partitioning tendency into this organic phase may be used to predict the enhancer potency. CS flux enhancement along the lipoidal pathway of HMS stratum corneum was determined with the 1‐alkyl‐2‐azacycloheptanones, 1‐alkyl‐2‐piperidinones, 1,2‐dihydroxypropyl decanoate, 1,2‐dihydroxypropyl octanoate, n‐alkyl‐β‐D‐glucopyranosides, 2‐(1‐alkyl)‐2‐methyl‐1,3‐dioxolanes, 1,2,3‐nonanetriol, and trans‐hydroxyproline‐N‐decanamide‐C‐ethylamide as enhancers. Enhancement factors (E values) were calculated from the permeability coefficient and solubility data over a range of E values. Comparisons of the enhancer potencies for all studied homologous series and the carbon number of the n‐alkyl group revealed a nearly semilogarithmic linear relationship with a slope of ∼0.55, which is consistent with the hydrophobic effect. Moreover, comparisons of the enhancer potencies of all the enhancers with the n‐hexanol–phosphate buffered saline (PBS), n‐octanol–PBS, n‐decanol–PBS, and n‐hexane–PBS partition coefficients showed very good correlations for the n‐alkanol solvents but not for n‐hexane. This result supports the interpretation that the enhancer potency is directly related to the ability of the enhancer molecule to translocate to a site of action via its free energy of transfer from the bulk aqueous phase to a semipolar microenvironment in the stratum corneum lipid lamella that is well mimicked by water‐saturated n‐alkanols. © 2003 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1305–1322, 2003

Mechanistic Study of Alkyl Azacycloheptanones as Skin Permeation Enhancers by Permeation and Partition Experiments with Hairless Mouse Skin

In previous studies (Yoneto et al., 1995. J Pharm Sci 84:312-317; Kim et al., 1992. Int J Pharm 80:17-31; and Warner et al., 2001. J Pharm Sci 90:1143-53), the transport enhancing effects of four homologous series of enhancers-the n-alkanols, 1-alkyl-2-pyrrolidones, 1,2-alkanediols, and N,N-dimethylalkanamides - on the transport of steroidal permeants across hairless mouse skin (HMS) were investigated. Isoenhancement concentrations are defined as the aqueous concentrations for which different enhancers induce the same extent of permeant transport enhancement, E, for the lipoidal pathway of the stratum corneum (SC). Our studies have shown that the E = 10 isoenhancement concentrations of these four homologous series were nearly the same when compared at the same n-alkyl group chain length and therefore that the contribution of the polar head group toward the enhancer potency was found to be essentially constant. In the present study, we have determined the isoenhancement concentrations (E = 10) for the 1-alkyl-2-azacycloheptanone series [1-butyl-2-azacycloheptanone (BAZ), 1-hexyl-2-azacycloheptanone (HAZ), and 1-octyl-2-azacycloheptanone (OAZ)] and compared the results with those of the previously studied four homologous series. We have found that the E = 10 isoenhancement concentrations (aqueous phase concentrations) of the 1-alkyl-2-azacycloheptanones (Azs) are around 10 times lower than those for the previously studied four homologous series when compared at the same alkyl group chain length. This indicates an approximately 10 times higher potency of Azs. This finding was a point of interest because the polar group of Azs is similar to that of 1-alkyl-2-pyrrolidones (Aps). To further probe the nature of the mechanism of action of the Azs and Aps and to better understand the lower E = 10 isoenhancement concentrations found with the Azs, it was decided (a) to determine the equilibrium partitioning (uptake) of the Azs and the Aps from the aqueous phase into the HMS SC at E = 10, and (b) to determine the equilibrium partitioning (uptake) of a surrogate permeant, estradiol (E2beta), into the SC in the absence of and in the presence of Azs and Aps at E = 10. The following were the outcomes from the two partitioning studies. Firstly, at the E = 10 isoenhancement concentrations, the extent of partitioning (uptake) of the Azs and Aps into the intercellular lipids of the HMS SC was found to be approximately the same, even though the E = 10 isoenhancement concentrations (aqueous phase concentrations) of the Aps were around 10 times greater than those of the Azs. We interpret this to mean (whereas the potencies of the Azs are around ten times greater than those of the Aps when related to their aqueous concentrations) that the potencies of the two enhancer series are about the same when expressed in terms of their concentrations in the intercellular lipid phase of the SC. Another outcome of the partitioning studies has been the finding that the extent of partitioning into the intercellular lipids of the SC at E = 10 isoenhancement conditions for both the Azs and Aps is essentially independent of the n-alkyl chain length (from butyl to octyl). A third result from these experiments has been that the partitioning of E2beta (the surrogate permeant) into the HMS SC under E = 10 isoenhancement concentration conditions is approximately the same with the Aps and Azs as enhancers. For both the Aps and Azs, the E2beta SC partitioning enhancement was found to be in the range of 5-6 at E = 10. This comparable partitioning enhancement for E2beta in the presence of Aps and Azs at E = 10 suggests that the same mechanism was involved and that these enhancers act, in part but to a significant extent, by inducing a higher partitioning tendency of the permeant into the transport rate-limiting lipoidal domains of the SC. (c) 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:297-310, 2003

Ammonia, the GABA neurotransmitter system, and hepatic encephalopathy.

There appears to be a consensus that hepatic encephalopathy (HE) is a metabolic encephalopathy with a multifactorial pathogenesis. One of the factors considered to be important in the pathogenesis of HE is ammonia. However, the mechanisms by which ammonia contributes to the manifestations of HE remain poorly defined. Ammonia could be more definitively implicated in the pathogenesis of HE if its effects can be shown to lead to an enhancement of inhibitory neurotransmission. In this context the effects of ammonia on the GABA (gamma-aminobutyric acid) neurotransmitter system may be relevant. Ammonia, at the modestly increased concentrations that commonly occur in precoma HE (0.15 mM-0.75 mM), has been shown to increase GABA-induced chloride current in cultured neurons, probably by modifying the affinity of the GABA(A) receptor for GABA. Comparable ammonia concentrations also enhanced synergistically the binding of a GABA agonist and a benzodiazepine (BZ) agonist to the GABA(A) receptor complex, phenomena which would enhance the neuroinhibitory effects of these ligands. Also, GABA increased the potency of ammonia-induced enhancement of the binding of a BZ agonist to the GABA(A) receptor complex, and brain levels of BZ agonists are elevated in liver failure. In addition, ammonia has been shown to inhibit astrocytic uptake of GABA by 30%-50%, an effect which would increase the synaptic availability of GABA at GABA(A) receptors. Furthermore, increased ammonia concentrations upregulate the peripheral-type benzodiazepine receptor in the outer membrane of astroglial mitochondria, thereby enhancing astrocytic mitochondrial synthesis and release of neurosteroids. Some neurosteroids, for example tetrahydroprogesterone (THP) and tetrahydrodeoxycorticosterone (THDOC), are potent agonists of the GABA(A) receptor complex, on which there are specific binding sites for neurosteroids, that are distinct from those for BZs and barbiturates. Tetrahydroprogesterone and tetrahydrodeoxycorticosterone levels were found to be increased in a mouse model of acute liver failure, and, when THP or THDOC was injected into normal mice, sedation and Alzheimer type II astrocytic changes in the cortex, striatum, and hypothalmus were induced. Each of these direct or indirect effects of ammonia on the GABA neurotransmitter system has the potential of increasing inhibitory neurotransmission, and, hence, contributing to the manifestations of HE.

Influences of alkyl group chain length and polar head group on chemical skin permeation enhancement

Previous investigations in our laboratory on the influence of the n‐alkanols and the 1‐alkyl‐2‐pyrrolidones as skin permeation enhancers for steroid molecules as permeants demonstrated that the enhancer potencies (based on aqueous concentration values) of these two homologous series were the same when compared at the same alkyl chain length; that is, the contribution of the hydroxyl group and that of the pyrrolidone group to enhancer potency were the same. The purpose of the present study was to further investigate what was believed to be a somewhat surprising finding, and two additional homologous series, the 1,2‐alkanediols and N,N‐dimethylalkanamides, were selected for study as enhancers. Corticosterone (CS) flux enhancement along the lipoidal pathway of hairless mouse skin stratum corneum was determined with 1,2‐hexane‐, 1,2‐octane‐, and 1,2‐decanediol and with N,N‐dimethylhexanamide, N,N‐ dimethylheptanamide, N,N‐dimethyloctanamide, and N,N‐dimethylnonanamide as enhancers. The enhancement factor (E) for the lipoidal pathway was calculated from the CS permeability coefficient and the CS solubility data over a 4 to 100 range of E values. Comparisons of the enhancer potencies of all four homologous series revealed that the enhancer potencies of all were very nearly the same when compared at equal alkyl group chain length. Moreover, the contribution of each of the polar head groups toward the enhancer potency was essentially constant, independent of the alkyl group chain length. It was reasoned that this outcome was either the result of the random selection of four polar head groups making the same contribution to enhancer potency or the result of these particular polar head groups not contributing to enhancer potency. To test the hypothesis that the former was more likely than the latter and that a suitable semipolar organic phase may mimic the microenvironment of the polar head group at the site of enhancer action, n‐octanol–phosphate buffered saline (PBS) and n‐hexane–PBS partition coefficients were determined for all the enhancers. The n‐octanol–PBS partition coefficients for the enhancers, but not the n‐hexane–PBS partition coefficients, were very nearly the same when compared at equal alkyl group chain lengths; this result supports the hypothesis that each of the four polar head groups likely contributes the same toward the enhancer potency and locates in the semipolar region of the hairless mouse skin stratum corneum lipid bilayers, which is well‐approximated by water‐saturated n‐octanol. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1143–1153, 2001

アンスラサイクリン系薬物の初代培養肝実質細胞からのグルコース分泌に与える影響と細胞傷害性

Anthracyclines have been used to cure a variety of cancers, and they are one of the most effective anti-cancer drugs. However, due to the simultaneous occurrence of serious side effects, successful chemotherapy is difficult to administer in clinical cancer treatment. Therefore, to evaluate the side effects, we examined the influence of anthracycline derivatives, especially epirubicin (EPI), on the glucose secretion and cytotoxicity of anthracycline derivatives in primary culture of rat hepatocytes.In this study, EPI reduced the spontaneous glucose secretion dose-dependently in a primary culture of rat hepatocytes. However, EPI enhanced the increased-secretion induced by glucagon, and the potency of such enhancement at low concentrations was stronger than that at high concentrations. In contrast, aclarubicin (ACL) reduced both the spontaneous glucose secretion and the increased-secretion. Furthermore, Anthracycline derivatives increased the lactate dehydrogenase (LDH) leakage both time- and dose-dependently, and the potency order was idarubicin≥ACL >daunorubicin> EPI. The degree of cytotoxicity as evaluated by measuring the glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels and by performing an MTT-assay was also similar to the results obtained for LDH leakage.These results suggest that EPI stimulates the sensitivity of rat hepatocytes to other endogenous agents, and this influence may be involved in the degree of cytotoxicity for anthracycline derivatives.

Enhancement of differentiation induction of mouse myelomonocytic leukemic cells by extracellular ATP.

We examined the effect of ATP on the terminal differentiation of mouse myelomonocytic leukemic M1 cells to macrophages. Although ATP alone did not induce M1 cell differentiation, addition of ATP with the differentiation inducer, interleukin 6 (IL-6), enhanced the induction of differentiation by IL-6 about two-fold. Comparison among several adenine nucleotides revealed that the order of effectiveness on differentiation enhancement was ATP > ADP > AMP > or = adenosine. Using reactive blue 2, a P2 receptor antagonist, we confirmed that the effect of ATP on the stimulation of differentiation was mediated through the P2 purinergic receptor. Examination of cytosolic [Ca2+] elevation by ATP and comparison of potency of differentiation enhancement among several ATP analogs demonstrated that the effect of differentiation enhancement was mediated through P2y purinergic receptors expressed on M1 cell surface. Within 3 h of exposure, ATP alone slightly increased the expression of differentiation-related immediate early response genes, c-myc and JunB, and ATP also enhanced the IL-6-induced expression of these genes. Induction of JunB expression by ATP analogs correlated with their potency of differentiation enhancement, which suggested that induction of JunB by ATP is one of signaling pathways involved in the exertion of its differentiation-enhancing effect.

Early postnatal treatment with muscimol transiently alters brain GABA A receptors and open-field behavior in rat

The long-term effects of treatment with muscimol, a GABA A agonist, and with ethanol, also shown to have a GABAergic profile of action, on subsequent behavior and on the binding parameters of GABA A receptors were studied in Wistar rats. Rats were given two daily injections of muscimol (0.1 mg/kg M1 group of 0.2 mg/kg M2 group) or ethanol (1.5 g/kg) between the 1st and 21st postnatal days, with saline-treated animals serving as controls. At the age of one month, the activity of the M2 rats was decreased in the open-field arena. At the age of four months, ethanol-treated rats consumed less 10% ethanol solution in a free choice situation. No changes were seen in the elevated plus-maze test. Maximal GABA stimulation of [ 3H]flunitrazepam binding was decreased in the cerebellum and hippocampus in M2 rats at the age of one month but not four months. A significant positive correlation was found in all rats between maximal GABA-stimulated [ 3H]flunitrazepam binding in the cerebellum and ambulation in the open-field arena at the age of one month. Ethanol intake correlated negatively with maximal GABA stimulation of [ 3H]flunitrazepam binding in the cerebral cortex. None of the treatments affected the cerebellar binding of an imidazobenzodiazepine, [ 3H]Ro 15-4513. There was an age-dependent decline in the efficacy and potency (EC 50) of the GABA enhancement of [ 3H]flunitrazepam binding in the cerebellum in all treatment groups. Basal binding of [ 3H]flunitrazepam to hippocampal and cerebellar membranes as well as binding of [ 3H]Ro 15-4513 to cerebellar membranes was also decreased as a function of age. The results suggest that muscimol exposure induces a transient change in GABA A receptor sensitivity. Also, open-field behavior, ethanol consumption and GABA A receptor mechanisms may be interrelated in the rat.

Absorption enhancement of rectally infused cefoxitin sodium by medium-chain fatty acids in conscious rats: concentration-effect relationship.

The objective of this study was to assess the relative absorption promoting potency in terms of concentration-effect relationships of the medium-chain fatty acids hexanoic acid, octanoic acid, decanoic acid, and dodecanoic acid in conscious rats, using cefoxitin sodium as the rectally delivered model compound. Rectal uptake of cefoxitin, which was absorbed to a limited extent without enhancer (30 +/- 25%), proved to be significantly enhanced by 2.0 M sodium hexanoate, 0.69 M sodium octanoate, and 0.22 M sodium decanoate, resulting in mean bioavailabilities of 102 +/- 24, 68 +/- 25, and 68 +/- 10%, respectively. Thus, increasing fatty acid chain length results in increased enhancing potency from hexanoic acid to decanoic acid. However, using dodecanoate a statistically significant effect could not be reached, because of its limited aqueous solubility. Optimal chain length for absorption enhancement by medium-chain fatty acids is probably determined by interplay of intrinsic effects on mucosal permeability and solubility of the medium-chain fatty acid.

Interaction Between Lymphocytes and Inflammatory Exudate Cells

Abstract Thymocytes of C3H/He mice were poorly triggered to induce DNA synthesis by phytohemagglutinin but fairly well triggered when they were mixed with small number of inflammatory peritoneal exudate polymorphonuclear leukocytes or macrophages, or with the soluble product(s) of these cells. Peritoneal exudate cells were mainly harvested at 3 hr, largely consisting of polymorphonuclear leukocytes, and at 96 hr, largely consisting of macrophages, after intraperitoneal injection of 0.2% sodium caseinate. Addition of 22 to 26 × 103 of 3-hr exudate cells, 22 to 24 × 103 of 48-hr exudate cells, or 22 to 24 × 103 of 96-hr exudate cells to syngeneic thymocyte culture (3 × 106) resulted in marked enhancement of DNA synthesis by thymocytes stimulated with 1 µ1 PHA. Similar enhancement of DNA synthesis was also observed in the presence of 200 µ1 of cellfree supernatant fluid of peritoneal exudates of 3 hr or 9 hr after casein stimulation. Conversely, two hundred microliters of 0-hr, 48-hr, or 96-hr peritoneal fluid showed a slight suppressing effect on thymocyte response and 50 to 100 µl of these exudates showed no enhancing effect. Furthermore, 50 to 800 µ1 of culture supernatants from 1.6 × 106 cells/4 ml of 3-hr exudate cells, 96-hr exudate cells, or purified macrophages also caused a similar enhancement of DNA synthesis by thymocytes. On the other hand, excess numbers of exudate cells (27 to 28 × 103 of 3-hr exudate cells, or 25 to 29 of 96-hr exudate cells) or excess amounts of culture supernatants (800 µl from 6.4 to 12.8 × 106/4 ml of 3-hr cells, or 200 µl from 12.8 × 106/4 ml, 400 µl from 6.4 × 106/4 ml, or 800 µl from 3.2 × 106/4 ml of 96-hr cells) diminished the enhancing potency on thymocytes. The cooperative function by polymorphonuclear leukocytes or by macrophages on the lymphocyte response was thus postulated.