Even if coupling of peptide fragments is not completed colospa 135 mg with amex, this kind of impurities is usually easily separated buy colospa 135mg otc. An important consideration during the development of the segment condensa- tion approach is the evaluation and design of the appropriate fragments buy colospa 135 mg on-line, taking into account their preparation frst and second, their condensation. Thus, the solubility of the fragments in condensation reactions has to be taken into account (an estimation or a prediction can be made but it is of no assurance without actual preparation). Fur- thermore, the potential epimerization during the condensation reaction is a common risk that should be avoided or reduced. Thus, Gly or Pro as C-terminal residues of the fragments is the priority if possible, in order to totally avoid racemization. Evaluation of the linker and the solid support is another cru- cial point to take into consideration. Side-chain-protecting groups have to be stable into the cleavage conditions of the peptide fragments of the projected peptide. Thus, Fmoc/tBu chemistry is commonly preferred, in combination with super acid-sensitive linkers/resins. Their development and commercial availability has allowed synthetic access to even longer peptide sequences. In the hybrid approach, the condensation of the protected fragments is carried out using the classical coupling reagents, which are described in Chapter 4 and Section 6. Two peptide fragments, one containing a C-terminal thioester and the other containing an N-terminal cysteine residue, are assembled by chemoselective coupling. Native chemical ligation is a reversible transthioesterifcation, followed by amide formation. Intramolecular nucleophilic attack of an α-amino group on the initial thioester product occurs only when the thiol is on the side chain of an N-terminal Cys, thus regenerating the thiol functional group of the Cys side chain and giving a fnal ligation product containing a native peptide bond at the site of ligation (Scheme 2. Initial thiol–thioester exchange step is fully reversible, whereas the second amide-forming step is irre- versible under the reaction conditions. Because of this, eventually, only the desired amide-containing product is formed, even in the presence of internal Cys residues in either peptide segment. This reversible-irreversible two step reaction mechanism is the essence of the native chemical ligation method. The main benefts of the native ligation strategy are the reduced solubility problems that commonly appear in the fragment-condensation strategy, and the absence of reagents that should be purifed. In this approach the side chain protecting groups of the fragments are removed before fragment ligation. The reaction takes place in an aqueous environment in neutral pH, in order to give a native peptide bond at the ligation point. Limitation of this hybrid technology is the mandatory use of a Cys residue at the N-terminal of the frst fragment, and the synthesis of an appropriate C-terminal thioester in the second fragment, which in some cases provides poor yields [197]. Side chain protection of the fragments is not essential, but favors the reaction in aqueous media. More interestingly, a side-chain-assisted chemical ligation has been reported lately, with no limits to the assembled amino acids [211]. Continuous improvement of ligation strategies provides an additional tool to peptide chemists to overcome more immediate challenges in view of the signifcantly increased demand for larger peptides. Cyclization offers many advantages, including increased proteolytic resistance and also enhanced biological activity, when compared to their linear counter- parts in some cases [215, 216]. Cyclic peptides can be classifed depending on their linkage: (a) head-to-tail type, when the N- and C-terminus are joined; (b) side-chain-to-C-terminal or N-terminal-to-side-chain, when a side-chain is linked to the C- or N-terminus; and (c) side-chain-to-side-chain, when two side-chains are joined. Linkage is usually an amide bond but can also be a disulfde or another type of functionality (Figure 2. Cyclizations in solution have the limitation that they must be carried out under high dilution conditions because of the risk of dimerization and oligomerization, and thus, high volumes of solvents are consumed. In contrast, in solid phase, and due to site iso- lation, intramolecular cyclization is favored. Furthermore, work-ups can be avoided since coupling reagents can be washed away, thereby releasing the fnal cyclized pep- tide in good purity. To obtain the most common head-to-tail cyclic peptides in the solid-phase, the N- and C-terminus must be free and not anchored to the resin. Side-chain anchoring approach: Here, the amino acid side-chain is linked to the solid support and the C- and N-terminus are orthogonally protected. Once chain elongation is fnished, the deprotection of both ends and subsequent cyclization and cleavage delivers the fnal cyclized product. Numerous amino acids have been used for side-chain anchoring, including Asx/Glx [220–226], Lys/Orn [220, 227], Ser/Thr [220, 228], Tyr [220, 224, 229], His [230, 231], and Cys [232, 233], on the usual supports and linkers for peptide synthesis. In a more general approach, the preformed handle can be coupled frst to the solid phase, and the C-terminal protected amino acid introduced by reductive amination. For the synthesis of cyclic peptides, the allyl ester of the corresponding amino acid is commonly used. After elongation of the peptide chain by standard Fmoc chem- istry, the cyclic peptide is constructed as described above for the side-chain anchoring approach. A major milestone was the synthesis of the antitumoral depsipeptide Kahalalide F [240] (Figure 2. The linear chain was then elongated until the introduction of 5-methylhexanoic acid and then the branch fnished by introducing dipeptide Alloc-Phe-Z-didehydroaminobutyric acid (Dhb), previously prepared in solution. After releasing the branched peptide from the resin, the fnal cyclization was performed in solution, giving the depsipeptide in good yields. The same strategy was also successfully applied to the synthesis of the natural product Triostin A [244]. For depsipeptides with simpler architectures, a protocol that allows automatic syn- thesis of the linear chain by Boc chemistry was developed [246]. The remaining couplings are performed using in situ neutralization protocols and capping steps. Polymeric forms of the desired peptide, especially in cyclic peptides, are also known. Much of what applies to the purifcation of synthetic peptides from synthetic procedure is also relevant to peptide isolation from complex biological material. Consequently, in any case, the purifcation of a crude peptide to homogeneity can hold great challenges; however it is an absolute prerequisite for successful and meaningful structural and functional analyses. In this context, it should be mentioned that all potential impurities probably could not be removed by a single chromatographic method, but rather by a combination of methods. This method is also known as gel permeation, molecular sieve, gel-exclusion, and size-exclusion chromatography. In this method, the chromato- graphic matrix consists of porous beads, and their size defnes the size of macro- molecules that may be fractionated. Proteins and/or peptides that are too large to enter the bead pores have less volume to pass through, and consequently elute frst from the column. Sequentially, smaller macromolecules that can insert some, but not all, of the pores are retained slightly longer in the matrix and exit the column next. Finally, small molecules flter through most of the pores, and they emerge from the column with an even larger elution volume. The main advantage of this method is that no binding of the sample is required, no harsh elution conditions are used, and thus, it rarely inacti- vates enzymes, and often is used as a signifcant step in peptide or protein purifcation. A limi- tation of the method is the slow separation of the macromolecules, mainly because the column should be narrow and long in order to achieve suffcient component separa- tion. The whole process can be accelerated by the use of pumps or more sophisticated high pressure chromatography equipment and/or matrices allowing faster fow rates, whereas this can hold higher risk for suffcient separation. Another crucial limitation is poor resolution given that in this method the peptide/protein does not bind to the matrix. For that reason, gel-fltration chromatography is used mainly as a separation tool when only a small amount of contaminants is present. In many cases, it is used for sample desalting or for changing the buffer of the sample [248]. In this method, selecting the appropriate ion exchange matrix is signifcant for sample separation. An anion-exchange matrix is derivatized with pos- itively charged groups, whereas cation exchange contains negatively charged groups.

Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs colospa 135mg without a prescription. Potent and selective inhibition of human immunodefciency virus type 1 transcription by piperazinyloxoquinoline derivatives generic colospa 135 mg without prescription. Long-term treatment with novel glycogen synthase kinase-3 inhibitor improves glucose homeostasis in ob/ob mice: molecular char- acterization in liver and muscle generic colospa 135 mg. Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse. Evidence of zeta protein kinase C involvement in polymor- phonuclear neutrophil integrin-dependent adhesion and chemotaxis. Docking sites on substrate proteins direct extracellular signal-regulated kinase to phosphorylate specifc residues. A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. Protein kinase C-epsilon is responsible for the protection of preconditioning in rabbit cardiomyocytes. C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo. Most people’s general impression of peptides is that they are large molecules that are easily digested by proteases. These two important draw- backs mean that peptides are often considered as being less stable with low oral bioavailability. Consequently, administration by injection is often required, and the peptides are readily inactivated by peptidases and cleared from the body. Moreover, they have a relatively higher risk of immunogenic effects when compared with small drug molecules. Owing to their relatively larger size and often hydrophilic nature, sol- ubilization, and the delivery of peptide drugs across membranes become challenges. Cost-wise, the synthesis of peptide drugs is often considered more expensive if one associates cost with molecular size. Viewed from the opposite side, where a detriment is reinterpreted as a beneft, pep- tide drugs hold several key advantages over small drug molecules. Therefore, peptide drugs are expected to be very potent because when compared to small drug molecules, peptide drugs have high activity and high specifcity due to very few unspecifc bindings to nontargeted sites. On the same line of reasoning, pep- tide drugs would have fewer drug–drug interactions, although, as previously stated, they have an increased risk of immunogenic effects. Considering that peptide drugs are large molecules composed mainly of natural amino acids with high target speci- fcity and are easily degraded by peptidases, peptide drugs would in theory exhibit lower toxicity than small drug molecules. Likewise, considering that peptide drugs have diffculty crossing membranes, they are less likely to accumulate in tissues and thus have a lower risk of adverse drug reactions over time. The sheer largeness of peptide drugs also means that they are more biologically and chemically diverse. In actual practice, however, peptide drugs are often used to derive small nonpep- tide drug molecules. Doing so offers the benefts from both classes and the fne line that differentiates between a peptide drug and small drug molecule becomes faded. Indeed, after a lengthy process of rational drug design where residues are changed from natural amino acids to nonnatural amino acids then to nonamino acids, it becomes rather challenging at times to classify if a drug is peptide or nonpeptide. Although we would like to classify a nonpeptide drug as a compound that does not possess any amino acid, out of respect for the developers of the drugs, in this chapter, we will keep the nonpeptide or peptide assignments that the drug developers have chosen, and will thus avoid any debate over semantics. We will focus on success- ful stories of peptide-derived drugs that are processed by enzymes. We will try to be as up-to-date as possible in the information that we provide at the time that this chapter is being written. It should be noted that, in this chapter, most comparisons done between differ- ent drugs are restricted to our own personal viewpoint; because of legal reasons and personal pride, the drug developers would claim originality to their own discoveries. Hence, we would like the readers to read with an open mind and come up with their own interpretations of the information that we provide. During the process of changing a peptide drug to a peptide-like drug and eventually to a nonpeptide drug, the naming of each residue becomes confusing because two or more residues may be merged into one functional structure. We will be using the Schechter and Berger [1] nomen- clature that assumes that the substrate binds to the active site of an enzyme in an extended backbone conformation. Within the active site, subsites, also referred to as pockets, ′ are denoted as Sn and Sn, where n represents the number of subsite away from the catalytic S1 subsite, with the prime symbol denoting the opposite direction. Often, N-terminal residues are referred as Pn, whereas C-terminal ′ residues are referred as Pn. The naming of peptide drugs follows the same rules as ′ ′ that of peptide substrates. For example, P2–P1–P1–P2 is a tetrapeptide drug with a ′ scissile bond between the P1 and P residues. For peptide inhibitors, the inhibitory 1 unit, which is the unit that prevents enzyme cleavage, is assigned to the P1 residue. One should keep in mind that because the numbering is based on the subsites of the active site rather than the sequential order of the residues of the peptide drug, and that the chemical structures of the enzyme and peptide drug are three-dimensional by nature, that in some cases, the numbering of the residues of the peptide drug may not follow a sequential order. In simpler words, there are cases where the peptide drug does not bind to the active site in an extended backbone conformation. An example of an irregular order numbering is argatroban, a direct thrombin inhibitor, which has aP3–P1–P2 sequence (Section 5. Hence, it is often easier to commercialize natural enzymes or activators of enzymes found in nature, and to develop inhibitors of enzymes, than to create more potent enzyme activators. A philosophical reasoning for this observation could be that nature has selected the best enzymes and their activators, whereas man can only copy or destroy nature’s refnements. Despite the previous statement, researchers have designed a few enzyme activators, such as α-methyldopa and droxidopa (Section 5. Here, we are loosely equating the term enzyme activator to substrate, because as far as we are aware, there is no allosteric activator in the pharmaceutical market. Most activators of enzymes, or the enzymes themselves, are developed via either extraction of pharmacologically active natural substances from a crude inexpensive natural source or by replicating the natural substances by synthetic means. On the contrary, most potent inhibitors of enzymes are derived from natural lead compounds, or from natural substrates that have been corrupted to become enzyme inhibitors. From our own experience, the frst step in substrate-based drug design of modula- tors is to establish an assaying system for enzyme activity. A modulator is either an activator or inhibitor, which in our case, applies to a substrate or its peptide inhibitor. As the initial step, a reproducible enzyme activity assay system must be developed from a substrate and enzyme that both must be stable and pure. It is noteworthy that the enzyme often can process several different substrates and the choice of substrate, especially in substrate-based design of enzyme inhibitors, will determine the structural outcome of the derived modulators. In order to improve the processing effciency of the substrate by the enzyme, the substrate and enzyme may be structurally altered by synthetic means to improve purity and stability, so as to reduce variations between experiment results. Often, the fnal substrate used in the assay is a shortened yet active version of a natural substrate, and the enzyme is modifed from its natural form to prevent self-digestion. Any drastic change from the natural substrate or enzyme could be viewed by the scientifc community as a huge leap from the substrate and the natural form of the enzyme, and thereby negatively refecting on the research as a false image of nature. A common method of substrate-based design of inhibitors entails the introduc- ′ tion of an inhibitory unit near the scissile bond, between the P1 and P1 residues of the substrate. The inhibitory unit is a modifed version of the P1 residue of the substrate such that the enzyme can recognize and bind to the inhibitory unit at the catalytic site, but the enzyme cannot readily cleave the inhibitor. A common mech- anistic feature of protease inhibitors is the presence of a transition state isostere, as a part of the inhibitory unit, to simulate the transition state of amide bond hydrol- ysis, as depicted in Figure 5. B from enzyme from enzyme Transition state mimetic inhibitor Pro N H O O O H H O O − O N N H O H O Figure 5. Our recent studies combined neutron diffraction crystallography to conclusively pro- vide direct experimental evidence of the catalytic mechanism of the protease and its inhibition by the inhibitory unit [5]. In the initial design of protease inhibitors, other than the central inhibitory unit, the remaining residues of the inhibitor are kept similar to that of the substrate.

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Research has demonstrated that parents are less likely to supervise video games cheap 135 mg colospa overnight delivery, compared to other forms of media discount colospa 135mg, suggesting there is potential for young people to ignore classifications purchase 135 mg colospa visa. Internet Content-control software is a form of software designed for controlling what content is permitted to a user on the internet. The restrictions on which web pages can be accessed can be applied at various levels. While research has not investigated the impact of these forms of software on reducing illicit drug use, they may represent useful tools in restricting exposure of drug influences to young people. British Medical Association (2006) Legalising illicit drugs: a signposting resource. Ministry of Health (1926) Report of the Departmental Committee on Morphine and Heroin Addiction (The Rolleston Report). World Health Organization (2007) International statistical classification of diseases and related health problems, 10th revision (2e). American Psychiatric Association (1994) Diagnostic and statistical manual of mental disorders (4e). Goldman D, Oroszi G & Ducci F (2005) The genetics of addictions: uncovering the genes. House of Commons Science and Technology Select Committee Drug classification: making a hash of it: fifth report of session 2005-2006. European Monitoring Centre for Drugs and Drug Addiction (2011) Annual report on the state of the drugs problem in Europe. Hoare J & Moon D (eds) (2010) Drug misuse declared: findings from the 2009/10 British Crime Survey. Home Office (2012) Drug misuse declared: findings from the 2011/2012 British Crime Survey. The Scottish Government (2012) 2010-11 Scottish crime and justice survey: drug use. Department of Justice (2010) Experience of drug misuse: findings from the 2008/09 Northern Ireland Crime Survey. Hay G, Gannon M, Casey J et al (2011) Estimates of the prevalence of opiate use and/or crack cocaine use, 2009/10: Sweep 6 report. Fuller E (2012) Smoking, drinking and drug use amongst young people in England 2011. Measham F, Moore K, Newcombe R et al (2010) Tweaking, bombing, dabbing and stockpiling: the emergence of mephedrone and the perversity of prohibition. Newcombe R (2004) Attitudes to drug policy and drug laws: a review of the international evidence. Royal Society for the encouragement of Arts, Manufactures and Commerce (2007) Drugs – facing facts. Bailey R, Fuller E & Ormston R (2010) Smoking, drinking and drugs: reaction to reform. Scottish Government (2010) Scottisocial attitudes survey 2009: public attitudes to drugs and drug use in Scotland. Roques B (1999) La dangerosité de drogues: rapport au Secrétariat d’Etat à la Santé. Best D, Gross S, Vingoe L et al (2003) Dangerousness of drugs: a guide to the risks and harms associated witubstance use. Rolles S & Measham F (2011) Questioning the method and utility of ranking drug harms in drug policy. Nutt D (2011) Let not the best be the enemy of the good: a reply to Caulkins et al. Room R (2011) Scales and blinkers, motes and beams: whose view is obstructed on drug scheduling? Darke S & Hall W (2003) Heroin overdose: research and evidence-based intervention. Darke S, Degenhardt L & Mattik R (2007) Mortality amongst illicit drug users: epidemiology, causes and intervention. O’Driscoll P, McGough J, Hogan H et al (2001) Predictors of accidental fatal drug overdose among a cohort of injection drug users. Warner-Smith M, Darke S, Lynskey M et al (2001) Heroin overdose: causes and consequences. Favrod-Coune T & Broers B (2010) The health effect of psychostimulants: a literature review. Singleton J, Degenhardt L, Hall W et al (2009) Mortality among amphetamine users: a systematic review of cohort studies. Srisurapanont M, Ali R, Marsden J et al (2003) Psychotic symptoms in methamphetamine psychotic in- patients. Aldington S, Harwood M, Cox B et al (2008) Cannabis use and risk of lung cancer: a case-control study. Hall W (2009) The adverse health effects of cannabis use: what are they, and what are their implications for policy? Kuepper R, Van Os J, Lieb R et al (2011) Continued cannabis use and risk of incidence and persistence of psychotic symptoms: 10 year follow-up cohort study. Advisory Council on the Misuse of Drugs (2008) Cannabis: classification and public health. Arseneault L, Cannon M, Witton J et al (2004) Causal association between cannabis and psychosis: examination of the evidence. Rubino T, Zamberletti E & Parolaro D (2012) Adolescent exposure to cannabis as a risk factor for psychiatric disorders. Macleod J, Oakes R, Copello A et al (2004) Psychological and social sequelae of cannabis and other illicit drug use by young people: a systematic review of longitudinal, general population studies. A scientific statement from the American Heart Association Acute Cardiac Care Committee of the Council on Clinical Cardiology. Darke S, Kaye S & Duflou J (2006) Comparative cardiac pathology among deaths due to cocaine toxicity, opioid toxicity and non-drug-related causes. Kaye S & Darke S (2004) Non-fatal cocaine overdose among injecting and non-injecting cocaine users in Sydney, Australia. Alaraj A, Wallace A, Mander N et al (2010) Effect of acute cocaine use on vasospasm and outcome in aneurysmal subarachnoid hemorrhage. Kaye S & Darke S (2004) Injecting and non-injecting cocaine use in Sydney, Australia: physical and psychological morbidity. European Monitoring Centre for Drugs and Drug Addiction (2007) Cocaine and crack cocaine: a growing public health issue. Darke S, Kaye S & Duflou J (2005) Cocaine related fatalities in New South Wales, Australia 1993-2002. Rogers G, Elston J, Garside R et al (2009) The harmful health effects of recreational ecstasy: a systematic review of observational evidence. Miotto K, Darakjian J, Basch J et al (2001) Gamma-hydroxybutyric acid: patterns of use, effects and withdrawal. Hickman M, Carnwath Z, Madden P et al (2003) Drug-related mortality and fatal overdose risk: pilot cohort study of heroin users recruited from specialist drug treatment sites in London. Smyth B, Hoffman V, Fan J et al (2007) Years of potential life lost among heroin addicts 33 years after treatment. Shahani R, Streutker C, Dickson B et al (2007) Ketamine-associated ulcerative cystitis: a new clinical entity. European Monitoring Centre for Drugs and Drug Addiction (2009) Polydrug use: patterns and responses. Cruts G, Buster M, Vicente J et al (2008) Estimating the total mortality among problem drug users. British Medical Association (2007) Fetal alcohol spectrum disorders – a guide for healthcare professionals. British Medical Association (2004) Smoking and reproductive life – the impact of smoking on sexual, reproductive and child health. Cole C, Jones L, McVeigh J et al (2011) Adulterants in illicit drugs: a review of empirical evidence. Department of Health (2002) Getting ahead of the curve: a strategy for combating infectious diseases (including other aspects of health protection). Aldington S, Williams M, Nowitz M et al (2007) Effects of cannabis on pulmonary structure, function and symptoms. Bancroft A, Wilson S, Cunningham-Burley S et al (2004) Parental drug and alcohol misuse.

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The channels can accommodate rapid movement of leukocytes and large proteins such as IgG and albumin; they are an important pathway of watery secretion from the blood network to the tissue purchase 135 mg colospa with amex. The lamina propria contains a blood supply buy cheap colospa 135mg online, a lymphatic drainage system cheap colospa 135mg otc, and a network of nerve fibers. It is through the blood vessels in the lamina propria that drugs can gain entry to the systemic circulation. Lymph drainage from the vagina takes place to the iliac sacral, gluteal, rectal, and inguinal lymphatic nodes. The changes are associated with aging (neonate, juvenile, adult and senescence), biphasic sexual cycling (follicular and luteal phases) and pregnancy. This proliferation of cells leads to an increase in epithelial thickness, as well as in the number of layers (Figure 11. A parallel increase in the number of intercellular junctions renders the epithelium more cohesive. The number of desmosomes increases approximately 10-fold from the early to late follicular phase. Luted phase During the luteal phase, desquamation (shedding) occurs on the superficial epithelial layer, extending as far as the intermediate cells. The vaginal surface loses its intact structure and the epithelium becomes loose and porous. This cyclic desquamation is preceded by loosening of intercellular grooves, as well as a pore- like widening of the intercellular channels. Rodents have an estrous cycle characterized by diestrous (Diest), proestrous, estrous (E) and metestrous phases (Figure 11. The cyclical changes in the epithelium of rodents are similar to the changes in human, i. The vaginal epithelium becomes extremely thin, cell boundaries in the surface are less distinct, the micro-ridges of the cells are dramatically reduced, and the vagina is often invaded with leukocytes. Naturally, this thinning of the epithelium leads to a substantial increase in the permeability of this tissue. Pregnancy During pregnancy the most marked change occurring in the vagina is increased vascularity and venous stasis, and the epithelial layer is greatly thickened. Following delivery, the vagina requires several weeks to reestablish its prepregnancy appearance. The vaginal fluid is composed of cervical fluid (the vagina receives approximately 2 g of mucus/day from the cervix) and also small amounts of the secretion from Bartholin’s glands in the vaginal wall. However, the bulk of fluid to the tissue, and the lumen of the tissue, comes via transudation of fluid (via the intercellular channels) from the very extensive vascular bed in the tissue. During the normal menstrual cycle, the amount of fluid increases at ovulation, by mixing with the uterine fluid, oviductal fluid, follicular fluid, and even peritoneal fluid. The vaginal secretions, which serve as a protective barrier for infections, contain a variety of antimicrobial substances including lysozyme, lactoferrin, fibronectin, polyamines such as spermine and secretory IgA. The fluids also contain carbohydrate from the epithelial glycogen, amino acids, aliphatic acids and proteins. The bioavailability of drugs administered via the vaginal route is dependent on both the effective dissolution of solid drug particles (if present) in the vaginal fluids prior to absorption and the degree of deactivation by enzymes present in the fluids. The physiological cyclical changes in the amount of vaginal fluids present in the vaginal cavity means that fluctuations in vaginal bioavailability can occur. This acidity plays a clinically important role in preventing the proliferation of pathogenic bacteria and there is a correlation between the pH of the vaginal secretions and the inhibition of chlamydial infections. Vaginal pH affects the degree of ionization of drugs, which can affect their absorption properties (see Section 1. Physiological changes in the pH of the vaginal fluids can also result in fluctuations in vaginal bioavailability. For example, using casein as a substrate, the proteolytic activity determined in a 10% homogenate of rat vaginal membrane was found to be less than that in the small intestine. The influence of the ovarian cycle on protease activity in the vagina has also been demonstrated. For example, the trypsin-like activity in rat vaginal smears was found to be maximal at proestrus. The activity of β-glucuronidase, acid phosphatase, alkaline phosphatase, and esterase all vary in the vaginal tissue of premenopausal and postmenopausal women. As described in general terms for the transepithelial absorption of drugs at any site (Section 1. In contrast, lipid-soluble drugs are usually absorbed transcellularly, by passive diffusion through the epithelium, down a concentration gradient according to Fick’s Law (Section 1. Drug diffusion rates correlate with their lipid/water diffusion coefficients and are inversely related to their molecular size (Section 1. However, these general observations do not take into account the cyclical changes in the vaginal epithelium, which exert profound effects on vaginal absorption, especially for hydrophilic compounds. The permeability coefficient for the vaginal membrane (P ) is equal to the sum of the permeability coefficientm through the lipid pathway (P ) and the pore pathway (P ):l p P =P +Pm l p For lipophilic drugs, the contribution of the pore pathway to transport is negligible and drug absorption occurs transcellularly, via passive diffusion through the epithelial cells. For example, it has been shown that increasing the chain length (increasing the lipophilicity) of aliphatic alcohols and carboxylic acids results in an increased rate of vaginal absorption. In contrast, for hydrophilic drugs, the pore pathway constitutes the major absorption pathway and this pathway is influenced by the physiological changes in the thickness of the vaginal epithelium and also in the number of intercellular pores and aqueous channels. As described earlier, in rodents, during proestrus and estrus, the epithelium is thick, tightly cohesive and contains a large number of intercellular junctions. However, the metestrous and diestrous phases are characterized by a thinning of the epithelium and a pore- like widening of the intercellular channels. As the vaginal epithelial membrane barrier becomes thin, loose and porous, the permeability is enhanced, particularly to hydrophilic substances. Thus even high molecular 280 weight hydrophilic drugs can be absorbed by the intercellular route during the metestrous and diestrous phases. Several examples of this phenomena are described below: Salicylic acid Vaginal absorption of salicylic acid in different pH buffers has been investigated in rats during proestrus and diestrus. For the unionized, lipophilic form of the drug, the rate of vaginal absorption is rapid and similar for both stages. The unionized, lipophilic form is absorbed via transcellular passive diffusion and thus not affected by the stage of the estrous cycle. However, for the ionized, water-soluble form, a significant difference in the degree of absorption is observed: • proestrus (tight epithelium)=29% absorbed; • diestrus (porous epithelium)=66% absorbed. The hydrophilic form is absorbed mainly through pore-like pathways such as the intercellular channels and thus is highly dependent on the stage of the cycle, with greater absorption occuring when the interceullular channels are wide and porous. The percentage of the dose of phenol red excreted in the urine increased more than an order of magnitude from the proestrous phase (2. Leuprorelin showed similar enhanced absorption during the permeable phase of the estrous cycle (Figure 11. Penicillin In humans high blood levels of penicillin, sufficient to be therapeutic, were demonstrated following insertion of a vaginal suppository near the end of the menstrual cycle and during menopause. In contrast, absorption was shown to be somewhat diminished during estrus and late pregnancy. Vidaribine The permeability coefficients of the hydrophilic antiviral compound vidaribine are 5 to 100 times higher during early diestrus or diestrus than during estrus. These results confirm that the cyclic changes in the reproductive system have profound implications for vaginal drug delivery as: • the vaginal permeability to hydrophilic substances is enhanced during the metestrous and diestrous stages of the estrous cycle, corresponding to the late luteal and early follicular phases of the menstrual cycle; • large fluctuations in absorption occur, depending on the particular stage of the menstrual cycle. Although it is well known that carrier-mediated transport systems exist for di- and tripeptides in the intestine, there is still no evidence for carrier-mediated transport of peptides across the vaginal mucosa, although prostaglandins have been demonstrated to utilize such a mechanism. Although there must be some type of endocytic transport of endogenous peptides into the epithelial cells in order to regulate proliferation, no receptor-mediated or bulk-fluid mechanisms have been reported. Hydrophilic compounds may be absorbed via the paracellular route, moving between the epithelial cells via passive diffusion whereas lipid soluble drugs are usually absorbed transcellularly, at rates which correlate with their lipid/water diffusion coefficients. However, in the vagina these factors must be considered in conjunction with the cyclical changes in the vaginal epithelium. Thus hydrophilic compounds show enhanced absorption during metestrus and diestrus, when the vaginal barrier becomes thin, loose and porous.

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