Introduction

Bone marrow transplantation is being used as a therapy for hematological disease and other disorders.¹ Unfortunately, a number of patients who receive allogeneic BMT suffer from chronic graft-versus-host disease (cGVHD) following the procedure.^{2, 3, 4, 5, 6} Oral symptoms in extensive form are present in approximately 80% of patients.⁵ Usually, atrophy of mucous membranes promotes the development of severe periodontitis and caries. Owing to the involvement of the salivary and lacrimal glands, the majority of patients with disseminated cGVHD develop sicca syndrome.⁷ In addition to the subjective complaints, patients with salivary gland dysfunction are susceptible to increased caries, oral pain, frequent infections and difficulties in speaking, chewing and swallowing. These difficulties may lead to inadequate nutrition and a decline in quality of life.⁸ This salivary flow reduction can be ameliorated by the administration of oral pilocarpine.³

The purpose of this study was to evaluate the unstimulated whole-salivary biochemical composition in cGVHD patients before and after the administration of oral pilocarpine.

Materials and methods

Patients and study design

Twenty-eight patients with cGVHD (as diagnosed by a physician), who were suffering from xerostomia with some remaining salivary function, were enrolled in a double-blind, placebo-controlled design study in the BMT Center of Sharyaty Hospital at Tehran University of Medical Sciences (TUMS) between August 2005 and August 2006.

Criteria for exclusion from the study were age, gender matching considerations and patients with cardiovascular diseases, uncontrolled asthma, angle closure glaucoma or hepatic impairment.

Thirteen patients were randomly assigned to case (eight men and five women, average age 33.5, range from 20 to 48 years) and 15 to control groups (10 men and 5 women, average age 31.6, range from 20 to 49 years). The case group was orally administered 5 mg of pilocarpine (Salagen, Novartis, UK), four times per day (20 mg/day) for 7 days. The control group received an identical-looking placebo.

The clinical physician and patients were unaware of the case and control drugs. The Ethics Committee of TUMS, Iran, approved the study protocol. Informed verbal and oral consent were obtained from each patient.

Saliva collection

Saliva was collected in the morning from each participant; thereby controlling any possible effects that circadian rhythm may produce in saliva composition. Unstimulated whole saliva was performed by the spitting method. All subjects refrained from eating and drinking for 1 h before the sampling. Each individual was seated for approximately 5 min, before 3–5 ml of unstimulated saliva was collected into a pre-weighed plastic cup over a period of about 25 min. The saliva-filled cups were weighed and the weight of the cup subtracted. The flow rate was calculated in g/min, which is almost equivalent to ml/min.⁹

Unstimulated whole saliva was collected four times for each patient. The first samples were collected 30 min before pilocarpine or placebo intake, and subsequent samples collected 1 h, 1 day and 1 week after drug administration. The saliva specimens were immediately stored at -70°C for later determination of saliva composition.

Saliva analysis

Whole saliva was assessed colorimetrically by a spectrophotometer and using affiliated kits (Pars Azmoon, Tehran, Iran) for the analysis of whole-saliva calcium, total protein and IgA concentrations. Total protein concentration was measured by the Biurt method,¹⁰ using bovine serum albumin as a standard; calcium by the Arsenozo reaction method,¹¹ sodium concentration was measured by flame photometry and IgA by immunoturbidometry.¹⁰ Saliva outputs of these parameters were calculated by multiplying flow rate and each saliva composition. Changes for all saliva parameters were calibrated from the primary saliva (base line).

Statistical analysis

Results were tested for statistical significance in the differences between case and control groups by un-paired Student's t-test. Differences among means were considered statistically significant if P<0.05. Data are expressed as means.e.m.

Results

There was a significant difference in flow rate between case and control groups in the second samples, taken 1 h after drug administration (Table 1); mean saliva flow rate of case was higher than control group. However, the mean difference of saliva flow rate between case and control groups in the other samples was not significant. Saliva flow rate changes were significant in the second samples, but were not significant in the third and fourth samples (Figure 1).

Figure 1.

Unstimulated whole-saliva changes of Na, Ca, total protein and IgA outputs and flow rate following administration of placebo or pilocarpine, 20 mg/day orally (5 mg four times a day). Each line represents the mean and s.e.m. BL=base line (30 min before treatment); 1 h, 1 d and 7 d=1 h, 1 day and 7 days elapsed following treatments, respectively. ^*Different from placebo, P<0.05.

Full figure and legend (82K)

Table 1 - Unstimulated whole-saliva flow rate and Na, Ca, total protein and IgA concentrations and their outputs following administration of placebo or pilocarpine, 20 mg/day orally (5 mg four times a day).

Full table

No significant differences were observed in mean Na, Ca, IgA and total protein concentrations and their changes between case and control groups at any sample point (Table 1 and Figure 2).

Figure 2.

Unstimulated whole-saliva changes of Na, Ca, total protein and IgA concentrations following administration of placebo or pilocarpine, 20 mg/day orally (5 mg four times a day). BL=base line (30 min before treatment); 1 h, 1 d and 7 d=1 h, 1 day and 7 days elapsed time following treatment, respectively. Each line represents the mean and s.e.m.

Full figure and legend (75K)

There was significant difference in mean salivary sodium and total protein outputs, and their output changes, between case and control groups in the second samples alone (Table 1 and Figure 1). Sodium and total protein outputs and their output changes in case group were higher than in control. However, no significant differences were found between case and control groups in Calcium and IgA outputs or their output changes at any sample point (Table 1 and Figure 1).

Patients who had received pilocarpine expressed satisfaction with their treatment. There was only one report of drug-related adverse effects – one patient claimed that his migraines had increased in intensity; in contrast, two patients claimed amelioration of their ocular dryness.

Discussion

Saliva is critical to the preservation and maintenance of oral health.^{8, 12, 13, 14, 15, 16, 17} Xerostomia is a major feature of cGVHD.^{6, 18, 19, 20} Temporary symptomatic relief can be offered by moistening agents and saliva substitutes, and is the only option for patients without residual salivary function. The carboxymethylcellulose-based artificial saliva does not replace the many salivary macromolecules critical to protective and other functions of saliva. In patients with residual salivary function, oral administration of pilocarpine is effective in increasing salivary flow and improving the symptoms of xerostomia.^{8, 21} Pilocarpine was found to be more effective than the artificial saliva.²² It has been shown that pilocarpine increases goblet cell numbers.²² Furthermore, more patients reported that it ameliorated their xerostomia, and more patients wanted to continue with it after the study.^{23, 24} However, pilocarpine was found to be associated with more side effects than the artificial saliva. These side effects were usually reported as being mild.^{23, 24} Some side effects, such as increasing lacrimal flow rate, are pleasant and can be considered as a benefit of pilocarpine in the treatment of xerostomia in cGVHD. In this study, there was only one drug-related adverse effect reported – one patient claimed an increase in migraines. In contrast, two patients had improvement in ocular dryness.

Our data indicated that there was no significant difference in flow rate of pilocarpine- and placebo-treated patients in the first, third and fourth samples, although flow rate of the case group in the third sample was higher than control group. But the flow rate and its changes in case group were significantly higher than control group in the second sample, 1 h after intake. In salivary gland hypofunction, the peak action of oral pilocarpine administration in Fox's report²⁵ was 30 min after intake, and afterward it decreased gradually. It seems that the second sampling was at the peak of pilocarpine action, because it had the most secretion, but the third and fourth samplings may not have been at the peak of drug action due to the variation in elapsed time after drug intake, when the samples were taken.

It has been shown that some saliva composition such as Na, IgA and total protein increased in cGVHD.¹⁹ Oral pilocarpine was able to reduce and normalize the elevated levels of Na, total protein and IgG salivary concentrations.¹⁹ In this study, no significant differences were observed in Na, Ca, IgA and total protein concentrations and their changes between case and control groups in any of the samples. There were also no significant differences in Ca and IgA outputs and their changes. However, a significant difference in Na and total protein outputs and their changes were observed in the second samples. The changes in Na, Ca, IgA and total protein outputs in the case group were higher than the control. It seems that pilocarpine may increase the output of saliva composition. Consistent with others^{3, 4, 19, 26} our study may indicate that pilocarpine normalizes salivary flow and composition alteration in cGVHD patients.

A significant statistical increase was observed only in the second sample; nevertheless, this elicited comments of satisfaction from patients after treatment. Any increase in salivation, no matter how small, which is accompanied by an increase in the production of beneficial constituents, may be a benefit to patients with xerostomia.^{3, 4}

References

1. Souza LN, Carneiro MA, de Azevedo WM, Gomez RS. Vascular endothelial growth factor (VEGF) and chronic graft-versus-host disease (cGVHD) in salivary glands of bone marrow transplant (BMT) recipients. J Oral Pathol Med 2004; 33: 13–16. | Article | PubMed | ChemPort |
2. Sullivan KM, Shulman HM, Storb R, Weiden PL, Witherspoon RP, McDonald GB et al. Chronic graft-versus-host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression. Blood 1981; 57: 267–276. | PubMed | ISI | ChemPort |
3. Singhal S, Mehta J, Rattenbury H, Treleaven J, Powles R. Oral pilocarpine hydrochloride for the treatment of refractory xerostomia associated with chronic graft-versus-host disease. Blood 1995; 85: 1147–1148. | PubMed | ChemPort |
4. Nagler RM, Nagler A. Pilocarpine hydrochloride relieves xerostomia in chronic graft-versus-host disease: a sialometrical study. Bone Marrow Transplant 1999; 23: 1007–1011. | Article | PubMed | ChemPort |
5. Alborghetti MR, Correa ME, Adam RL, Metze K, Coracin FL, de Souza CA et al. Late effects of chronic graft-vs. host disease in minor salivary glands. J Oral Pathol Med 2005; 34: 486–493. | Article | PubMed | ISI | ChemPort |
6. Soares AB, Faria PR, Magna LA, Correa MEP, de Sousa CA, Almeida OP et al. Chronic GVHD in minor salivary glands and oral mucosa: histopathological and immunohistochemical evaluation of 25 patients. J Oral Pathol Med 2005; 34: 368–373. | Article | PubMed | ChemPort |
7. Schaefer UW, Beelen DW. Bone Marrow Transplantation, 2nd edn. Karger Basel: Germany, 1996, pp 86–89.
8. Fox PC, van der Ven PF, Baum BJ, Mandel ID. Pilocarpine for the treatment of xerostomia associated with salivary gland dysfunction. Oral Surg Oral Med Oral Pathol 1986; 61: 243–248. | Article | PubMed | ChemPort |
9. Navazesh M. Methods for collecting saliva. Ann NY Acad Sci 1993; 694: 72–77. | Article | PubMed | ChemPort |
10. Thomas L. Clinical Laboratory Diagnostics, 1st edn. TH-Books verlagsgesellschaft: Frankfurt, 1998, pp 644–647, 241–247, 231–241, 131–137, 664–678.
11. Gindler EM, King JD. Rapid colorimetric determination of calcium in biologic fluids with methylthymol blue. Am J Clin Pathol 1972; 58: 376–382. | PubMed | ChemPort |
12. Mandel ID. The functions of saliva. J Dent Res 1987; 66: 623–627. | PubMed |
13. Mandel ID. The role of saliva in maintaining oral homeostasis. J Am Dent Assoc 1989; 119: 298–304. | PubMed | ChemPort |
14. Slomiany B, Murty VL, Piotrowski J, Slomiany A. Salivary mucins in oral mucosal defense. Gen Pharmacol 1996; 27: 761–771. | PubMed | ISI | ChemPort |
15. Humphrey SP, Williamson RT. A review of saliva: normal composition, flow, and function. J Prosthet Dent 2001; 85: 162–169. | Article | PubMed | ChemPort |
16. Navazesh M. Dry mouth: aging and oral health. Compend Contin Educ Dent 2002; 23: 41–48. | PubMed |
17. Narhi TO. Prevalence of subjective feelings of dry mouth in the elderly. J Dent Res 1994; 73: 20–25. | PubMed | ChemPort |
18. Levy S, Nagler A, Okon S, Marmary Y. Parotid salivary gland dysfunction in chronic graft-versus-host disease (cGVHD): a longitudinal study in a mouse model. Bone Marrow Transplant 2000; 25: 1073–1078. | Article | PubMed | ISI | ChemPort |
19. Nagler RM, Nagler A. Sialometrical and sialochemical analysis of patients with chronic graft-versus-host disease – a prolonged study. Cancer Invest 2003; 21: 34–40. | Article | PubMed | ChemPort |
20. Nagler R, Barness-Hadar L, Lieba M, Nagler A. Salivary antioxidant capacity in graft versus host disease. Cancer Invest 2006; 24: 269–277. | Article | PubMed | ChemPort |
21. Guchelaar HJ, Vermes A, Meerwaldt JH. Radiation-induced xerostomia: pathophysiology, clinical course and supportive treatment. Support Care Cancer 1997; 5: 281–288. | Article | PubMed | ChemPort |
22. Aragona P, Di Pietro R, Spinella R, Mobrici M. Conjunctival epithelium improvement after systemic pilocarpine in patients with Sjogren's syndrome. Br J Ophthalmol 2006; 90: 166–170. | Article | PubMed | ChemPort |
23. Davies AN, Singer J. A comparison of artificial saliva and pilocarpine in radiation-induced xerostomia. J Laryngol Otol 1994; 108: 663–665. | PubMed | ChemPort |
24. Davies AN, Daniels C, Pugh R, Sharma K. A comparison of artificial saliva and pilocarpine in the management of xerostomia in patients with advanced cancer. Palliat Med 1998; 12: 105–111. | Article | PubMed | ChemPort |
25. Fox PC. Systemic therapy of salivary gland hypofunction. J Dent Res 1987; 66: 689–692. | PubMed |
26. Singhal S, Powles R, Treleaven J, Rattenbury H, Mehta J. Pilocarpine hydrochloride for symptomatic relief of xerostomia due to chronic graft-versus-host disease or total-body irradiation after bone-marrow transplantation for hematologic malignancies. Leuk Lymphoma 1997; 24: 539–543. | PubMed | ChemPort |