the functions of the salivary glands are controlled by the autonomic nervous system and influenced by the sensory nervous system .
when parasympathetic impulses dominate , salivary flow is greatly enhanced and the saliva has a low protein content .
studies of animal and human innervation have revealed that parasympathetic nerve fibers are present around acinar cells , ducts , and blood vessels in the major salivary glands .
a research has also shown that beside the classic transmitters noradrenaline and acetylcholine , neuropeptides such as substance p ( sp ) , calcitonin gene - related peptide ( cgrp ) , and vasoactive intestinal polypeptide ( vip ) ( figure 1 ) are present in the nerve fibers of the autonomic nervous system as well as in the auriculotemporal nerve , facial nerve , and cervical dorsal root fibers .
these neuropeptides are known to cause salivation in rats [ 27 ] . in recent years
, the mechanisms of actions of drugs that used to treat xerostomia have been elucidated pharmacologically from the viewpoint of salivary neuropeptide levels .
anethole trithione and pilocarpine have been shown to elevate sp and cgrp levels in human saliva [ 811 ] .
cevimeline hydrochloride hydrate ( cevimeline ) ( figure 2 ) is a novel muscarinic acetylcholine receptor agonist currently being developed as a therapeutic agent for sjgren 's syndrome .
sjgren 's syndrome is a serious and chronic autoimmune disorder characterized by inflammation in the exocrine glands such as the salivary and lacrimal glands , leading to xerostomia ( dry mouth ) and xerophthalmia ( dry eyes ) .
cevimeline acts as a stimulator of the m3 acetylcholine receptor expressed on salivary glands and has been shown to increase saliva secretion in patients with sjgren 's syndrome .
although cevimeline is useful for the treatment of dry mouth , it only enhances saliva production in 60% of the patients , and the mechanism of the drug response is still unknown .
it is possible that individual variability of neuropeptide nerve stimulation in response to cevimeline may be involved in the variable drug response to cevimeline .
the objective of the present study is to examine the effects of cevimeline on saliva and plasma levels of sp- , cgrp- , and vip - like immunoreactive substances ( iss ) in humans , as markers of nerve stimulation of these neuropeptides .
cevimeline hydrochloride hydrate ( saligren capsule 30  mg ) was purchased from nippon kayaku co. ltd .
lactose ( merck hoei co. ltd . , osaka , japan ) was used as placebo .
synthetic human sp , cgrp and its fragment ( 837 ) , and vip were purchased from peptide institute , inc .
vip fragment ( 1128 ) was supplied by professor yajima ( kyoto university , kyoto , japan ) .
substance p antiserum ( y150 ) was purchased from yanaihara institute ( shizuoka , japan ) , cgrp antiserum ( 14160 ) from peptide institute , inc . , and vip antiserum ( t-4116 ) from peninsula laboratories ( california , usa ) .
seven healthy nonsmoking male volunteers aged 2431 ( median 27 ) years and weighing 5670 ( median 64 ) kg participated in this study .
all subjects had no history of xerostomia , and their baseline fasting salivary and plasma levels of sp- , cgrp- , and vip - is were within the normal ranges for healthy subjects reported previously [ 811 , 15 , 16 ] .
each subject gave informed consent after receiving explanation on the scientific purpose of the study .
the subjects fasted for at least 2 hours before the study was commenced and during the experiments .
we performed an open - labeled , crossover study between may and october 2010 . in each subject ,
cevimeline and placebo were studied in random order , in a crossover manner with an interval of one month between the two studies . on the day of study ,
all subjects finished lunch ( standardized lunch of less than 800  kcal ) before 12:00 .
each study was conducted from 14:00 to 18:00 in a room with temperature controlled at 25c , during which the subjects maintained a resting and relaxed state .
a single dose of cevimeline 30  mg ( cevimeline group ) or placebo ( placebo group ) was administered orally with 100  ml water . at scheduled times after the test drug was administered , saliva production was measured , and saliva samples were collected for assaying salivary neuropeptide levels , while blood samples were collected for measuring plasma neuropeptide levels . the dose of cevimeline in this study was the normal daily dose used in clinical therapy .
saliva and venous blood samples were collected before and at 30 , 60 , 90 , 120 , 180 , and 240  min after administration of cevimeline or placebo .
the volume of saliva produced in 5  min was measured by the saxon test , an oral equivalent of the schirmer test .
14 , kawamoto houtai zairyou , osaka , japan ) and a polyethylene pouch were weighed . after swallowing to remove any existing oral fluid
, saliva was collected by placing the two cotton balls onto the vestibule of the mouth for exactly 5  min .
the weight of saliva was determined by subtracting the original weight of the pouch and cotton balls from the weight obtained after the cotton balls were placed in the mouth .
the weight of the liquid was taken to be the salivary volume ( ml ) produced in 5 minutes .
unstimulated whole saliva specimens were collected by the spitting method according to navazesh and christensen .
the subjects rinsed their mouth thoroughly with deionized water and rested for a few minutes before saliva collection began .
after one minute practice collection , which was discarded , subsequently 3  ml of saliva was collected into a test tube containing 500 kallikrein inhibitor units / ml of aprotinin and 1.2  mg / ml of edta .
blood samples were collected into chilled tubes containing 500 kallikrein inhibitor units / ml of aprotinin and 1.2  mg / ml of edta .
the saliva samples were diluted 1  :  1 with 4% acetate buffer ( ph 4.0 ) , centrifuged at 3500  rpm for 5  min at 4c , and then the supernatant was diluted 2  :  3 with 4% acetate buffer ( ph 4.0 ) and loaded onto c18 reverse - phase cartridges ( sep - pak c18 ; millipore corp . , milford , ma , usa ) .
blood samples were centrifuged , and the plasma samples were diluted 1  :  4 with 4% acetate buffer ( ph 4.0 ) and loaded onto c18 reverse - phase cartridges .
after washing with 4% acetate buffer , neuropeptides in the columns were eluted with 70% acetonitrile in 0.5% acetate buffer ( ph 4.0 ) .
eluates were concentrated by spin - vacuum evaporation , lyophilized , and stored at 40c until use .
the recovery of sp- , cgrp- , and vip - is in saliva and plasma was greater than 90% using this extraction procedure [ 1921 ] .
neuropeptide levels in saliva and plasma were measured using highly sensitive enzyme immunoassays for sp , cgrp , and vip   as described previously .
an immunoplate ( nunc - immuno module maxisorp f8 , intermed , denmark ) coated with anti - rabbit igg ( 55641 , icn pharmaceuticals , inc . ,
human sp , cgrp fragment ( 837 ) , or vip fragment ( 1128 ) was conjugated with -d - galactosidase by n-(-maleimido - caproyloxy)-succinimide according to the methods of kitagawa et al . .
the enzyme immunoassays were specific and highly sensitive , with detection limits of 0.08 , 0.40 , and 1.00  fmol / well for sp- , cgrp- , and vip - is , respectively .
total release of each neuropeptide or saliva was calculated as the area under the level  or volume  time curve ( auc0240 ) using the trapezoidal method .
differences in neuropeptide - is level , salivary volume , and their auc0240 between the cevimeline and placebo groups were analyzed by paired t - test or mann - whitney u test .
the relationship between auc0240 of neuropeptide - is level and auc0240 of salivary volume was analyzed by pearson 's product - moment correlation coefficient .
statistical analyses were performed using the spss software package ( version 17.0 ; spss inc . , il , usa ) .
the salivary sp - is level - time profile and total release of sp - is ( auc0240 ) after a single oral dose of cevimeline or placebo are shown in figure 3(a ) and table 1 .
oral administration of cevimeline resulted in significant increases in salivary sp - is level at 30 , 60 , 90 , and 120  min ( 7.5  3.4 , 19.1  15.1 , 12.5  5.1 , and 9.9  4.1  pg / ml , resp . ) compared with the corresponding levels after placebo administration ( 4.0  1.5 , 5.2  1.8 , 5.6  2.4 , and 5.3  2.7  pg / ml ) .
furthermore , auc0240 was significantly higher after cevimeline administration ( 2420.9  744.6  pgmin / ml ) compared with placebo ( 1185.8  398.6  pgmin / ml ) .
on the other hand , no significant changes in salivary cgrp- and vip - is levels and auc0240 were observed after the administration of cevimeline ( figures 3(b ) and 3(c ) and table 1 ) compared with placebo .
the plasma sp- , cgrp- , and vip - is level - time profiles and total releases of sp- , cgrp- , and vip - is ( auc0240 ) after a single oral dose of cevimeline or placebo are shown in figure 4 and table 2
. cevimeline administration did not alter the plasma levels or auc0240 of sp- , cgrp- , or vip - is compared with placebo .
the changes in salivary volume and total release of saliva ( auc0240 ) after cevimeline or placebo administration are shown in figure 5 and table 3 .
cevimeline administration resulted in significant increases in salivary volume at 90 , 180 , and 240  min ( 5.6  2.8 , 5.7  1.8 , and 5.1  1.2  ml , resp . ) compared with the corresponding levels after placebo administration ( 3.4  1.3 , 3.4  1.5 , and 3.2  1.6  ml ) .
the auc0240 was also significantly higher after cevimeline administration ( 1200.8  403.4  mlmin ) compared with placebo ( 804.9  369.8  mlmin ) .
the relationship between auc0240 of sp - is level and salivary volume after administration of cevimeline or placebo is shown in figure 6 .
a significant correlation was observed between auc0240 of sp - is level and auc0240 salivary volume ( r = 0.55 , p = 0.042 ) .
in this study , we investigated the effects of cevimeline on saliva and plasma levels of sp- , cgrp- , and vip - is in healthy subjects .
past studies have established that salivary and plasma levels of sp- , cgrp- , and vip - is vary within 30  min after a meal and then maintain constant from 1 hour after a meal [ 8 , 9 , 1921 ] .
furthermore , it is known that the absorption of cevimeline is little affected by a meal .
these data support our study design , and the present study appropriately evaluates the effects of cevimeline on neuropeptide levels and saliva production without being affected by a meal .
sp is mainly localized in submandibular and parotid glands and increases blood flow via its vasodilatory effect in salivary glands , stimulates the production of saliva and amylase , and influences ionic flow in rats [ 23 , 24 ] .
previous report indicates that cgrp also enhances the release of saliva and amylase in rats [ 3 , 6 ] , and vip induces alterations in salivary fluid and protein secretion [ 4 , 25 ] . in the present study ,
a single oral dose of cevimeline resulted in significant increases in salivary sp - is level at 30 , 60 , 90 , and 120  min and in the auc0240 of sp - is compared with placebo administration , whereas cevimeline did not alter the plasma levels or auc0240 of sp - is .
anethole trithione and pilocarpine have also been reported to increase sp - is in saliva but not in plasma [ 811 ] .
these results indicate a close association of sp with the enhancement of salivary secretion by cevimeline , in the same manner as anethole trithione and pilocarpine .
in addition , these findings suggest that cevimeline may mainly promote salivary secretion from submandibular and parotid glands by increasing sp . on the other hand , no significant changes in salivary and plasma levels and auc0240 of cgrp- and vip
these findings suggest that pathways via cgrp and vip nerves may not be involved in the stimulatory effect on salivation by cevimeline . on the other hand ,
anethole trithione and pilocarpine increase not only sp but also cgrp levels in human saliva [ 811 ] . cevimeline acts as a selective stimulator of the m3 acetylcholine receptor expressed on salivary glands , and this selectivity may reflect the specificity of the cevimeline action on sp nerves in salivary glands .
oral cevimeline administration resulted in significant increases in salivary volumes at 90 , 180 , and 240  min and in the auc0240 compared with placebo administration .
furthermore , a significant correlation was observed between auc0240 of sp - is level and auc0240 of salivary volume , suggesting the possible involvement of sp in the cevimeline - enhanced saliva secretory activity .
a lag time was observed between elevation of salivary sp level and increase in salivary volume , suggesting that sp secreted from the sp nerves stimulated by cevimeline may initially increase blood flow and cause vasodilatation in salivary glands , followed by a gradual increase in salivary production .
however , some reports have suggested that human salivary glands are thought to lack an sp innervation of the acinar cells , and in vitro pieces of human submandibular glands do not respond with fluid secretion to the administration of sp , as judged by the release of potassium [ 26 , 27 ] .
furthermore , other neuropeptides not tested in this study may also be involved in the mechanism of enhancement of salivary secretion by cevimeline . therefore , this notion requires verification by further studies .
cevimeline is known to enhance saliva production in only 60% of the treated patients , and the mechanism of drug response remains unknown .
the present study shows a possibility that individual variability of sp nerve stimulation in response to cevimeline may account for the variable drug response to cevimeline , although it is uncertain whether this trend in healthy volunteers is also observed in patients .
therefore , further studies are required to investigate the effects of cevimeline in patients with conditions such as xerostomia .
this study demonstrated the effects of cevimeline on salivary and plasma levels of neuropeptides in humans .
a single oral dose of cevimeline resulted in a significant increase in salivary but not plasma sp - is level , and a significant correlation was observed between the total release of salivary sp - is and of salivary volume .
these findings suggest a close association of sp with the enhancement of salivary secretion by cevimeline .
a large - scale controlled study evaluating multiple dosing conditions of cevimeline would help to better understand the effects of cevimeline .
 cevimeline is a novel muscarinic acetylcholine receptor agonist currently being developed as a therapeutic agent for xerostomia . 
 we examined the effects of cevimeline on salivary and plasma levels of substance - p- ( sp- ) , calcitonin - gene - related - peptide- ( cgrp- ) , and vasoactive - intestinal - polypeptide- ( vip- ) like immunoreactive substances ( iss ) in humans . 
 an open - labeled crossover study was conducted on seven healthy volunteers . 
 saliva volume was measured , and saliva and venous blood samples were collected before and 30240  min after a single oral dose of cevimeline or placebo . 
 salivary and plasma levels of sp- , cgrp- , and vip - is were measured using a highly sensitive enzyme immunoassay . 
 a single oral dose of cevimeline resulted in significant increases in salivary but not plasma sp - is level compared to placebo . 
 cevimeline administration did not alter the salivary or plasma levels of cgrp - is or vip - is compared to placebo . 
 significant increases in salivary volume were observed after cevimeline administration compared to placebo . 
 a significant correlation was observed between the total release of sp - is and that of salivary volume . 
 these findings suggest an association of sp with the enhancement of salivary secretion by cevimeline . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions