Novel drug delivery of dual acting prodrugs of hydroxychloroquine with aryl acetic acid NSAIDs: Design, kinetics and pharmacological study

Drug Deliv. and Transl. Res.

DOI 10.1007/s13346-017-0420-5

ORIGINAL ARTICLE

Novel drug delivery of dual acting prodrugs

of hydroxychloroquine with aryl acetic acid NSAIDs: Design,

kinetics and pharmacological study

1

Joshi Poorvashree & Dhaneshwar Suneela

#

Controlled Release Society 2017

Abstract Rheumatoid arthritis (RA) is a chronic autoimmune

disease characterized by restricted movements of joints of

hand, feet, elbow, knees and neck but principally the synovial

joints. Though etiopathology is not exactly known, treatment

paradigms are evolving to provide a tighter control over symp-

toms and disease progression. Current trend is introduction of

disease modifying anti-rheumatoid drugs (DMARDs) at early

stages. Hydroxychloroquine (HCQ) and nonsteroidal anti-

inflammatory drugs (NSAIDs) are two mechanistically differ-

ent categories widely used in the management of RA where

the first arrests the disease progression while the latter offers

symptomatic relief. Present work aims at minimizing prob-

lems of slow onset and accumulation of HCQ in non-

targeted sites and local gastric intolerance to NSAIDs by de-

signing their mutual ester prodrugs. Synthesis of prodrugs was

achieved by CDI coupling and structures were confirmed by

and anti-inflammatory activities using standard animal

models. Among all, prodrugs of HCQ with licofelone (HL)

and aceclofenac (HA) produced superior analgesia, improved

weight gain, normalization of joint diameter/paw volume than

HCQ and physical mixtures of HCQ and NSAIDs.

Hematological and biochemical studies indicated significant

step up in RBC, Hb, platelet count, total protein nutrient

(TPN) levels and step down in WBC, serum glutamic-

oxaloacetic transaminase (SGOT) and serum glutamic-

pyruvic transaminase (SGPT) by the treatment with HL and

HA. Through these novel codrugs, problems of slow onset

and accumulation of HCQ in non-targeted sites and local gas-

tric intolerance to NSAIDs were well addressed. These dual

acting mutual prodrugs of two mechanistically different anti-

arthritic agents could be explored further as promising strate-

gy for effective management of RA.

1

13

IR, H–NMR, C–NMR, mass spectroscopy and elemental

analysis. Prodrugs resisted hydrolysis in acidic environment

of the stomach but exhibited significant release in small intes-

tine. Upon oral administration of prodrugs to rats, 40.5–49%

HCQ and 53.4–66.8% of NSAIDs were recovered in 8.5–10 h

in blood. Urine and feces samples pooled over a period of 24 h

exhibited 2.3–3.5% and 0.75–0.9% of HCQ, respectively,

without any presence of intact prodrugs or NSAIDs.

Prodrugs were pharmacologically evaluated for analgesic

.

Keywords Prodrugs Hydroxychloroquine NSAIDs

.

Arthritis Disease modifying agent CDI coupling

Accumulation

Abbreviations

HC

Healthy control

AAC

AC

Acetic acid control

Arthritic control

Electronic supplementary material The online version of this article

HCQ

Hydroxychloroquine

(doi:10.1007/s13346-017-0420-5) contains supplementary material,

HBA, HIN, HA, HD, HL Prodrugs of HCQ with biphenyl

acetic acid, indomethacin,

which is available to authorized users.

aceclofenac, diclofenac and

licofelone, respectively

HCQ + BPA, HCQ + Indo Physical mixtures of HCQ with

*

Dhaneshwar Suneela

suneeladhaneshwar@rediffmail.com

1

HCQ + Aceclo, HCQ +

Diclo, HCQ + Lico

biphenyl acetic acid, indometha-

cin, aceclofenac, diclofenac and

licofelone, respectively

Department of Pharmaceutical Chemistry, Poona College of

Pharmacy, Bharati Vidyapeeth University, Erandwane,

Pune, Maharashtra 411 038, India

Drug Deliv. and Transl. Res.

DMARDs

Disease modifying anti- rheu-

matic drugs

use of disease modifying anti- rheumatoid drugs

(DMARDs). But it provided poor long-term outcomes [4].

Out of the intense need for paradigm change in the therapy

of RA, past two decades saw focused efforts being directed

towards replacing traditional therapies through various trials

like COBRA (Combinatie Therapie Bij Rheamatoide

Arthritis), step up, step-down approach, TEAR (treatment of

early aggressive RA) study and GUEPARD trial. COBRA

trial achieved short-term clinical efficacy but provided long-

term structural benefits in early RA as compared to monother-

apy. Step-down strategy involves early and intensive treat-

ment with DMARD monotherapy which leads to overtreat-

ment. In step-down approach, there is tight control over RA by

using DMARDs like methotrexate. Step-up combination ther-

apy provided better clinical and functional outcomes. TEAR

study involved triple therapy which when compared with step-

up approach showed similar effectiveness [4, 5]. All these

evolving therapies provide benefit of controlling disease ac-

tivity as well as symptomatic relief through combination ther-

apy offering complete and long-term remission. The clinical

trials conducted so far to design an ideal therapy for RA con-

cluded into combination therapy of DMARDs to provide safe

intensive therapy with lower extent of adverse effects as com-

pared to monotherapy [6]. DMARDs slowly alter the course

and progression of RA while symptomatic, rapid acting

NSAIDs only suppress inflammation and decrease pain with-

out acting on the underlying cause of the disease [7]. For

getting a dual benefit, DMARDs can be prescribed in combi-

nation with NSAIDs [6].

Hydroxychloroquine (HCQ) is a slow acting DMARD,

generally used as a second line therapy for RA [8]. This anti-

malarial is used as a remission-inducing drug and has shown

broad compatibility with other antiarthritic drugs with superi-

or benefit to risk ratio so is generally recommended for sup-

pression of autoimmune disorders like RA, systemic lupus

erythematous and other connective tissue disorders. HCQ

blocks Bantigen processing^ in macrophages and presentation

of antigen-MHC complex to CD4+ cells which inhibits for-

mation of complex between antigenic peptide present on the

surface of macrophages with CD4+. Due to this, release of

inflammatory cytokines gets downregulated. HCQ acts on

Toll-like receptors (TLRs) which are responsible for initiating

immune responses and slow down progression of disease [9].

At physiological pH, HCQ mainly exists in a dicationic

form and readily gets trapped in tissues resulting in tissue

depot effect. Interestingly, its redistribution back into blood

is slow that results in delayed onset of antirheumatic effect

[10]. Therefore, high dose of HCQ is required for rapid

achievement of a steady state. The loading dose of HCQ in

humans is 400–600 mg while 200–400 mg is the maintenance

dose [11]. But higher dose of HCQ leads to higher extent of

dose-dependent side effects such as retinopathy which is

mainly due to its accumulation in cornea [12].

CDI

1,1′-Carbonyldiimidazole

Red blood corpuscles

White blood corpuscles

Serum glutamic-oxaloacetic

transaminase

Serum glutamic-pyruvic

transaminase

Complete Freund’s Adjuvant

Carboxymethyl cellulose

RBC

WBC

SGOT

SGPT

CFA

CMC

Introduction

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune

disorder characterized by restriction in movement of joints of

hand, feet, elbow knees, and neck. Patients with RA suffer

from worst quality of life. Inflammation of synovium, swell-

ing and pain in and around the joints are the hallmarks of RA.

If severity of inflammation is not checked in time, it may have

damaging effect on cartilage, elastic tissue and even the bone.

As the disease progresses loss of cartilage, narrowing of joint

space between bones, looseness of joints, deformity and loss

of mobility become very evident. Unfortunately joint defor-

mity is irreversible because of which doctors emphasize on

early diagnosis and aggressive therapy.

Exact cause of the disease is unknown but HLA-DRB-14

and PTPN22 genes are implicated in genetic susceptibility

towards the disease. HLA class II genes (HLA-DRB-14) locat-

ed in major histocompatibility complex (MHC) undergo struc-

tural polymorphism in the process of antigen presentation

which affects binding and presentation of peptides to auto-

reactive T cells. Similarly, gene encoding the intracellular

phosphatase protein tyrosine phosphatase non-receptor type

2

2 (PTPN22) has been shown to possess the second strongest

association with RA. The C1885T polymorphism of the

PTPN22 gene leading to an amino acid change from arginine

to tryptophan at amino acid position 620 activates body’s im-

mune system and confers risk for RA [1]. Some environmen-

tal factors and infectious agents also contribute towards RA.

This variable and unpredictable course of disease leads to joint

destruction, deformity and loss of function leading to residual

disability [2].

Recent trends recommend early recognition, diagnosis and

initiation of treatment for controlling inflammatory process,

joint damage, remission and restoration of normal physical

functions. Current treatment modalities focus on combination

therapy as well as use of biological modifiers [3].

In 1980s, a pyramid approach was used for treatment of RA

that was also referred to as Bgo low, go slow approach^. It

involved use of nonsteroidal anti-inflammatory drugs

(

NSAIDs) until development of joint damage followed by

Drug Deliv. and Transl. Res.

1

NSAIDs are frequently used as first line treatment for RA

to provide symptomatic relief [13]. Gastrointestinal side ef-

fects are very common with the long-term use of NSAIDs.

The main reason behind local irritation to gastric mucosa is

free carboxylic acid present in NSAID’s structure and trapping

mechanism. Currently, NSAIDs are combined with

gastroprotective agents for arthritic treatment.

synthesized prodrugs were recorded in CDCl using H–

3

NMR Varian Mercury 400 MHz with super conducting

magnet using TMS as internal standard at Sophisticated

Analytical Instrumentation Facility, Punjab (India).

Chemical shifts are reported in ppm downfield on δ scale.

The mass spectra were recorded employing electrospray

ionization technique on G6460A Triple Quad LC/MS/MS

System (Agilent Technologies, USA) and elemental anal-

yses of synthesized prodrugs was performed on Elementar

(Vario MICRO CUBE, Germany) at Poona College of

Pharmacy, Pune (India). In vitro and in vivo release studies

were carried out on Jasco PU model 2080 with UV detector

UV-Vis 2070 HPLC using Thermo C₁₈column (Hypersil

gold, 250 × 4.36 mm, 5 μ) (Mumbai, India). Institutional

Animal Ethical Committee’s approved experimental proto-

cols were followed for pharmacological screening of syn-

thesized prodrugs which was carried out with at the

CPCSEA-approved animal facilities of Poona College of

Pharmacy, Pune, India (Reg. No.100/1999/CPCSEA).The

animals were procured from National Toxicological

Centre, Pune India, for the present study. Acetic acid

(0.6%) used for analgesic activity was purchased from

Merck limited (Mumbai, India). Carrageenan (C-3889

Type IV) and Complete Freund’s Adjuvant (CFA) were

purchased from Sigma Chemicals (USA). Biochemical, he-

matological and histopathological evaluation was carried

out at Sai Pathology Labs, Pune, India. The tissue sections

were stained with hematoxyline-eosin, and images were

captured with Camera (Nikon optical microscope Eclipse

E-200 with resolution of 40X). Radiological screening was

carried out at Bhagwat X-ray Clinic, Pune, India on full

body X-ray machine.

In the present work, we propose a chemical formula-

tion or latentiation of a disease modifying agent (HCQ)

with aryl acetic acid class of NSAIDs into dual acting

mutual prodrugs or codrug offering disease modifying as

well as analgesic/anti-inflammatory effects for the effec-

tive management of RA. Our research group has reported

synthesis and in vitro kinetics of mutual prodrugs of HCQ

with ibuprofen and naproxen [14]. Continuing with the

same concept, we report here synthesis, in vitro/in vivo

release kinetics and extensive pharmacological screening

of five codrugs of HCQ and NSAIDs with objectives of

minimizing accumulation of HCQ in non-targeted tissues

in order to lower extent of associated side effects and

lowering local irritant action of NSAIDs on gastric muco-

sa for safe management of RA through their dual action.

By temporarily masking free carboxylic group of NSAIDs

with HCQ, local irritation of gastric mucosa can be min-

imized. Chemically conjugating two synergistic drugs

may additionally improve their delivery properties,

achieving better absorption as compared to coadministra-

tion in separate dosage form or as a combination prepara-

tion [15].

Materials and methods

4

-Biphenyl acetic acid and 1, 1′-carbonyldiimidazole

Synthesis of prodrugs

(

CDI) were purchased from Sigma Aldrich (USA). HCQ

and licofelone were generously gifted by IPCA

Laboratories Ltd. (Mumbai, India) and Enaltec

Laboratories ( Mumbai, I ndia), r espectively.

Indomethacin, diclofenac and aceclofenac were procured

as gift samples from Zim Laboratories (Nagpur, India).

All other chemicals used in the syntheses were either of

AR grade or synthetic grade which were purified before

use. Reactions were monitored by thin layer chromatogra-

phy performed on pre-coated silica gel plates-60 F264

Desulfation of HCQ sulfate to HCQ was carried out

with the help of 30% ammonia. Mutual prodrugs of

HCQ with NSAIDs were synthesized by CDI coupling

reaction at 10 °C. The scheme of synthesis is illustrated

in Fig. 1.

Conversion of HCQ sulfate into HCQ [16]

(

Merck, Germany) using DCM: n-hexane: TEA

0.8:0.2:0.05; v/v/v) solvent system. The TLC spots were

To solution of HCQ sulfate (500 mg, 1.15 mmol) in chlo-

roform (10 ml), 30% ammonia was added till solution

turned alkaline (pH 9) and was stirred for an hour at room

temperature. HCQ was extracted in chloroform. Organic

phase was separated, washed with water and dried over

sodium sulfate. Organic phase was evaporated on

located by exposing to iodine vapors or UV light. Reaction

solvent was evaporated by Rotavac (KikaWerke, HB4,

Germany). Melting points were recorded on programmable

melting point apparatus (Veego, India) and are uncorrect-

ed. The IR spectra of the synthesized compounds were

recorded on Jasco V-530 FTIR 4100 in anhydrous IR grade

Rotavac to get pure HCQ. M.P. 94 °C, R 0.6 (methanol:

f

ethyl acetate: TEA; 0.9:0.1:0.05 drops; v/v/v), percentage

yield 90%

1

3

potassium bromide. Proton and C- NMR of the

Drug Deliv. and Transl. Res.

OH

phosphate buffer (pH 7.4) in the range of 10–100 μg/ml.

Release study was performed in HCl buffer (pH 1.2) and

stomach homogenates till 3 h and in phosphate buffer (pH

7.4) and intestinal homogenates till 7 h, respectively.

Prodrug (10 mg) was introduced in 100 ml of either

0.05 M HCl (pH 1.2) or 0.05 M phosphate buffer (pH

H

N

H

N

a

O

R

Cl

b

Cl

O

R=

7

3

.4) in a beaker kept in constant temperature bath at

7 ± 1 °C with occasional stirring. Aliquots (5 ml) were

H

C

2

O

Cl

NH

withdrawn and replaced with the fresh aqueous buffer at

regular intervals of 0, 15, 30, 45, 60 min and every

N

Cl

O

3

0 min interval till 3 h for HCl buffer and 7 h for phos-

HBA Cl

HIN

phate buffer and 50 μl sample reconstituted with mobile

phase was injected in the column.

Cl

HA

HL

Cl

NH

N

In vivo release studies

Cl

HD

In vivo behavior of orally administered prodrugs was investi-

gated in Wistar rats (200–250 g; n = 3) housed in metabolic

cages individually under normal conditions (temperature

Fig. 1 Synthesis of prodrugs (HBA, HIN, HA, HD and HL). Reaction

conditions. a CDI coupling in CHCl b Reflux for 12–24 h

3

27 ± 0.5 °C, relative humidity 70 ± 0.5%, 12:12 h dark/light

cycle). HPLC methods were developed for simultaneous esti-

mation of HCQ, prodrugs and NSAIDs to study there in vivo

behavior. Same HPLC system, column and mobile phase were

used for this purpose as mentioned for in vitro release study.

All the kinetic studies were carried out in triplicate. Male

Wistar rats were starved for 24 h prior to use and were admin-

istered water ad libitum. Blood (0.5 ml) was withdrawn by

retro-orbital puncture and the reading was considered as zero

min reading. A suspension of each prodrug in 1.0 ml of phys-

iological saline was administered to the animals (Table 1).

Blood samples were collected in EDTA-coated tubes at an

interval of 15 min for the first 1 h. Then subsequently blood

was collected on a bi-hourly basis till 10th h and finally at 24th

h. The samples were analyzed by HPLC, using procedure

described above.

Synthesis of mutual ester prodrugs of HCQ with NSAIDs

To a solution of NSAIDs (2.98 mmol) in anhydrous chlo-

roform (10 ml), CDI (3.57 mmol) was added at room

temperature and was stirred for 2–5 h. HCQ (2.98 mmol)

in anhydrous chloroform (10 mL) was then added and

refluxed at 70 °C for 10–12 h. The reaction was moni-

tored by TLC using DCM: n-hexane: TEA (0.8.0.2:0.05;

v/v/v). The solution was concentrated using Rotavac and

the residue was dissolved in chloroform (10 ml) and sub-

sequently washed with 1 M sodium carbonate solution

(

×

2 × 10 ml), 10% HCl (2 × 10 ml) and distilled water (3

10 ml). The organic layer was separated, dried over

anhydrous sodium sulfate, evaporated and recrystallized

from ethanol. Further purification was achieved by col-

umn chromatography [silica gel (mesh size 60–120);

Merck, Germany for column chromatography packed in

n-hexane] using ethyl acetate: hexane (80:20; v/v).

Biological evaluation

Animals

In vitro release studies

Wistar rats (female, 150–200 g) were used for evaluation

of anti-inflammatory activity by carrageenan-induced rat

paw edema model and Freund’s adjuvant arthritis model.

Swiss albino mice of either sex (20–25 g) were used for

analgesic activity. The animals were distributed into 18

groups, having six animals each. Animals were housed in

solid bottom polypropylene cages and maintained at

24 ± 1 °C, with relative humidity of 45–55% and 12:12 h

dark/light cycle. Acclimatization period was 2 weeks. The

animals had free access to food (standard chow pellets,

Chakan Oil Mills, Sangli) and water, ad libitum. The doses

of prodrugs were calculated on an equimolar basis to HCQ

and are mentioned in Table 1. The standard and test

To ensure bioreversibility of synthesized prodrugs,

in vitro stability and hydrolysis kinetics were performed

in aqueous buffers of varied pH range and tissue homog-

enates of upper GIT of rats. Samples were analyzed in

triplicate and methods were validated as per ICH guide-

lines. Hydrolyzed products and prodrugs were simulta-

neously estimated by reverse phase HPLC using phos-

phate buffer (0.1 M, pH 3.0 ± 0.1): acetonitrile (55:45

v/v) at a flow rate of 0.8 ml/min on Thermo C18 column

at 240 nm. Calibration curves of prodrugs, HCQ and

NSAIDs were constructed in HCl buffer (pH 1.2) and

Drug Deliv. and Transl. Res.

Table 1 Change in paw volume in carrageenan-induced rat hind paw edema

a

Groups

Dose (mg/kg) 1h

3h

6h

Change in paw

volume

% Inhibition of

edema

Change in paw

volume

% Inhibition of

edema

Change in paw

volume

% Inhibition of

edema

HC

0.9 % saline

1ml)

0.0±0.0

--

0.0±0.0

--

0.0±0.0

--

(

#

AC

0.13±0.05

0.10±0.10

0.10±0.20

0.09±0.06

0.09±0.01

0.10±0.04

0.05±0.12

0.10±0.20

0.12±0.06

0.08±0.12

0.12±0.06

0.08±0.19

0.12±0.00

--

0.16±0.07

--

1.19±0.08

--

ns

**

ns

HCQ

HBA

HIN

HA

12

19

23.0

21.4

31.8

33.5

25.2

64.9

19.8

10.5

39.0

5.4

0.08±0.02

53.1

70.5

69.3

55.7

79.5

84.0

76.0

75.0

91.0

96.5

92.7

70.1

93.1

0.12±0.01

90

***

**

0.05±0.03

0.01±0.09

99.4

97.1

100

99.6

100

99.5

99.0

100

***

27.2

24

0.05±0.09

-0.01±0.01

**

***

0.07±0.02

-0.01±0.01

***

HD

22

0.03±0.11

-0.00±0.02

***

HL

25

0.03±0.07

-0.01±0.01

***

BPA

12

0.04±0.00

-0.01±0.01

***

Indomethacin 5

Aceclofenac 10

0.01±0.07

0.01±0.03

***

0.01±0.17

-0.02±0.01

***

Diclofenac

Licofelone

HCQ+BPA

HCQ+Indo

25

0.01±0.08

-0.02±0.05

***

5

41.2

9.3

0.01±0.03

-0.02±0.01

***

19+12

21.2+5

24+10

0.05±0.14

0.01±0.03

ns

***

0.12±0.005

9.3

0.01±0.06

0.01±0.01

ns

***

HCQ+

Aceclo

HCQ+Diclo 22+25

0.11±0.10

16.9

0.01±0.19

0.00±0.03

ns

**

***

0.13±0.02

0.11±0.11

3.2

0.07±0.09

58.9

59.3

0.01±0.07

99.2

99.5

ns

**

***

HCQ+Lico 25+5

17.6

0.07±0.03

0.01±0.00

#

a: Average of six readings ± SEM, statistically analyzed by one way ANOVA followed by Dunnett's Multiple Comparison test, P<0.01as compared to

healthy control, *P<0.05, **P < 0.01, ***P < 0.001, ns: non-significant as compared to AC.

compounds were administered orally as a suspension in

% sodium CMC.

Analgesic activity by acetic acid-induced writhing method

1

Acetic acid (0.6%) was injected (0.25 ml) intraperitoneally to

Swiss albino mice. Suspension of standard and test com-

pounds was administered prior to administration of acetic ac-

id. The mice were placed individually into glass chamber and

Statistical analysis

An average of six readings was calculated and data were

expressed as mean ± SEM. Statistical differences between

the groups were calculated by one-way ANOVA followed

by Dunnett’s Multiple Comparison test for carrageenan-

induced rat paw edema, analgesic activity and ulcerogenic

potential, and two-way ANOVA followed by Bonferroni’s test

for acetic acid- induced analgesic model, carrageenan- in-

duced rat paw edema model and Freund’s adjuvant arthritis

model. Statistical significance was considered at P < 0.05, P <

5

min were allowed to elapse. The mice were then observed

for a period of 10 min and the number of writhes was recorded

for each animal. For scoring purpose, a writhe is indicated by

stretching of the abdomen with simultaneous stretching of at

least one hind limb [17]. Percentage inhibition of writhes was

calculated by following formula:

C‐D

%

Analgesic activity ¼

Â 100

C

0

.01 and P < 0.001 when compared to arthritic control.

where,

C = average writhes in the control group

D = average writhes in the drug-treated group

Acute toxicity studies

Acute toxicity studies were carried out as per Organization for

Economic Co-operation and Development (OECD) guideline

No. 420 using stair case method. In this, animals in each group

received 2000 mg/kg dose of standard and test compounds.

After dosing, animals were kept under observations for 48 h

for abnormality and mortality.

Anti-inflammatory activity by carrageenan-induced rat paw

edema method

UGO BASILE Plethysmometer 7140, Italy, was utilized for

recording paw volume. The animals showing ± 0.5 variations

Drug Deliv. and Transl. Res.

in paw volume were selected. Carboxymethyl cellulose (CMC)

solution (1%; 0.5 ml) was administered to control group as

vehicle. Suspensions of test compounds in 1% CMC were pre-

pared and volume to be administered was calculated as per doses

mentioned in Table 1. Carrageenan (1%, 0.1 ml) was injected

into the sub-plantar portion of right hind paw, 1 h after admin-

istration of drugs. The volumes of injected and non-injected left

paws were measured at 1h, 3h, 4h and 6 h and differences in paw

volume of both the paws were calculated (before and after car-

rageenan administration). The following formula was used for

calculation of % inhibition [18].

method [20]. The livers were dissected, homogenized with

phosphate buffer (pH 7.4) and centrifuged (2 cycles of

15 min each at 10,000 rpm). The supernatant was filtered

through membrane filter (0.45 μm) and estimated by HPLC

for HCQ content [21, 22]. At the end of CFA-induced model,

all the animals were sacrificed and their stomachs were re-

moved and analyzed for ulcers. The ulcers were scored ac-

cording to the method reported by Cioli et al. and the ulcer

index was determined [23].

Results

ꢀ

ꢁ

Vt

Vc

Chemistry

%

Inhibition of edema ¼ 1‐

Â 100

Ester prodrugs of HCQ and five different aryl acetic acid

NSAIDs (biphenylacetic acid, indomethacin, aceclofenac,

diclofenac, and licofelone) were synthesized (HBA, HIN,

HA, HD, and HL, respectively) (Fig. 1) by reported

procedure [24]. Reactions were monitored by TLC

where, V = difference in paw volume before and after carra-

geenan injection of test animal

V = difference in paw volume before and after carrageenan

injection of carrageenan control animal.

t

c

(

DCM: n-hexane: TEA, 0.8:0.2:0.01; v/v/v). Synthesized

1

13

prodrugs were characterized by FTIR, H–NMR, C–

NMR, elemental analysis and mass spectroscopy. The

data of physico-chemical characterization of synthesized

prodrugs is provided in supplementary material. IR for es-

ter prodrugs showed stretching at 1730–1750 cm of

C = O group of ester, absence of C = O/–OH stretching

Anti-arthritic activity by Freund’s adjuvant-induced arthritis

model

−

1

Arthritis was induced by a single intra-dermal injection of

.1 ml of CFA into the sub-plantar region of the foot pad of

0

-

1

the left hind paw rats. Tuberculin syringe (1 ml) with 26 gauge

needle was used to administer the Freund’s adjuvant. The rats

were anesthetized with light ether inhalation prior to and dur-

ing adjuvant injection. The animals were housed in cages to

allow the development of full arthritis up to 12 days. The

dosing was started from the 12th day by oral route (Table 1)

and continued till the 28th day. The progression of arthritis

was evaluated by measuring change in joint diameter (Vernier

Caliper), change in paw volume (UGO Basile

Plehysmometer, Italy), change in body weight (weighing bal-

ance, Unitek, India) on 0th, 4th, 8th, 12th, 16th, 20th, 24th,

and 28th day after adjuvant injection. The blood samples were

withdrawn on the 28th day for biochemical assays (ALT/

SGPT, AST/SGOT, total protein, and C-reactive protein) and

complete blood count by retro-orbital puncture. Samples were

centrifuged at 10000 rpm at 4 °C for 10 min. Serum was

separated and assayed. After sacrificing animals, paws were

sent for histopathology of rat ankle joint. Sections of ankle

joint were stained by hematoxyline and eosin.

Photomicrographs were taken on Nikon optical microscope

Eclipse E-200 with resolution of 40X. Variables or tissue re-

actions like inflammatory infiltrates, synovial hyperplasia,

pannus formation and cartilage erosion were scored as per

standard method of grading of arthritis [19]. Radiological

evaluation was carried out and visual scoring was performed

and visual scoring values were calculated according to scoring

of carboxylic acid (1720–1706 and 3300–2500 cm re-

spectively) as well as –OH of stretching of HCQ (3700-

-

1

3584 cm ). The H–NMR spectra of ester prodrugs

showed absence of chemical shifts for protons of carbox-

ylic and aliphatic –OH and presence of chemical shift of

proton of secondary amine of HCQ backbone at δ 5.2–5.9.

1

3

The results of C–NMR, elemental analyses of the syn-

thesized prodrugs were in accordance with the calculated

values and were within the permissible % of variance

(0.4%). Molecular ion peaks observed for the synthesized

prodrug matched with anticipated molecular weights of

prodrugs. Partition coefficients and aqueous solubilities

of standard drugs and synthesized conjugates were deter-

mined experimentally. LogP_o_c_tof HCQ was found to be

3.87 while LogP_o_c_tvalues of NSAIDs were in the range

of 1.9–3.7. Conjugation of HCQ with NSAIDs enhanced

the partition coefficients of prodrugs in the range of 3.99–

4.90. The results of partition coefficients of prodrugs were

in accordance with their determined aqueous solubilities

(0.17–0.34 mg/ml).

In vitro release kinetics in aqueous buffers and tissue

homogenates of rats

New HPLC methods were developed for simultaneous esti-

mation of prodrugs in the presence of their released active

Drug Deliv. and Transl. Res.

Table 2 In vitro release kinetics of prodrugs in aqueous buffers, the stomach, and intestinal homogenates

Prodrug Incubation medium

HCl buffer (pH 1.2)/

stomach homogenates

Phosphate buffer (pH 7.4)

K ± S.D.

Intestinal homogenates

t

1/2

% Prodrug

%

K ± S.D.

t

1/2

% Prodrug

%

−1 a

(min )

(min)

hydrolyzed HCQ released (min )

(min)

hydrolyzed HCQ released

4.5 × 10⁻

2.6 × 10⁻

5.0 × 10⁻

3.4 × 10⁻

2.2 × 10⁻

3

153.30 87.50

265.10 70.00

137.50 90.20

198.30 80.00

55.30

34.80

45.10

43.80

30.60

5.4 × 10⁻³126.45 92.00

5.1 × 10⁻³135.88 88.60

58.15

44.03

48.11

50.35

32.70

HBA

HIN

HA

Stable

7.5 × 10⁻³

5.4 × 10⁻³126.34 92.00

2.5 × 10⁻³277.2

68.00

92.4

96.93

HD

HL

315.0

63.50

a

Average of three readings; follows first order kinetics

metabolites, i.e., HCQ and NSAIDs. Percentage of hydro-

lyzed prodrugs and release of HCQ and NSAIDs were calcu-

lated using equations generated from calibration curves.

Kinetic parameters like half-lives, rate constants and order of

kinetics of hydrolysis were also calculated (Table 2). No

change in the peak of prodrugs was noted in 0.05 M HCl

buffer (pH 1.2) and stomach homogenates of rats. Peaks of

prodrugs started decreasing from 2 to 3 h and completely

disappeared at 7 h in phosphate buffer and intestinal homog-

enates. Presence of HCQ and respective NSAIDs was ob-

served at 1.5–3 h.

Analgesic activity by acetic acid-induced writhing method

Synthesized prodrugs were screened for analgesic activity by

acetic acid-induced method where inhibition of writhes was

calculated and compared with standard groups. Table 3 shows

analgesic activity of standard and test compounds. Analgesic

effect of HCQ was non-significant at 60 min which was sig-

nificantly evident at 180 min owing to its slow onset of action.

Early onset of analgesic activity (at 60 min) was observed for

HA, HL and HIN with HA offering maximum analgesic ac-

tivity (42%). The standard NSAIDs; aceclofenac (63%),

licofelone (71%), diclofenac (66%), and indomethacin

(

61%) also produced significant analgesic effect with an early

In vivo release kinetics

onset of 60 min. Similarly physical mixture of HCQ with

aceclofenac (24%), diclofenac (31%) and licofelone (29%)

also exhibited significant analgesic effect at 60 min. From

To study in vivo behavior, equimolar doses of prodrugs were

orally administered (Table 1) to the rats kept in metabolic

cages. The blood samples were withdrawn by the retro-

orbital puncture at pre-determined time intervals. These bio-

logical samples were estimated by HPLC using newly devel-

oped and validated method. All prodrugs appeared in blood

between 1 and 1.5 h after oral administration with consistent

rise in concentration till 2.5–3 h. At 3 h, HCQ and NSAIDs

started appearing in blood with declining concentration of

prodrug in blood. At 8–9.5 h prodrugs completely disap-

peared. Urine/feces samples were collected at various time

intervals and pooled together over a period of 24 h in which

peaks for NSAIDs and prodrugs were not observed but small

quantity of HCQ was noted.

1

20 min onwards, there was a consistent rise in analgesic

activity in all groups treated with test and standard com-

pounds. Initially, (after 60 min of administration) advantage

of standard NSAIDs over the prodrugs was evident but after

1

80 min, equimolar doses of prodrugs produced significantly

better analgesic activity over NSAIDs (Table 3). At 240 min,

all groups exhibited 97–100% inhibition of writhes except

HIN (72%).

Anti-inflammatory activity by carrageenan-induced rat paw

edema

Change in paw volume of Wistar rats and percentage of inhi-

bition of edema were assessed by plethysmometer in

carrageenan-induced rat hind paw edema model. Results are

shown in Table 1. Healthy control group did not show any

change in paw volume. After administration of carrageenan,

significant increase in paw volume of arthritic control was

observed. The standard and test compounds were adminis-

tered to rats 1 h before the administration of carrageenan.

HCQ is reported to have slow onset of action; therefore,

Biological evaluation

Acute oral toxicity study

None of the animals of any group showed any behavioral

abnormality or mortality after administration of 2000 mg/kg

dose, during the observation period of 48 h.

Drug Deliv. and Transl. Res.

Table 3 Analgesic activity by acetic acid-induced writhing method

Groups

60 min

% inhibition 120 min

% inhibition 180 min

% inhibition 240 min

% inhibition

AA

74.33 ± 6.80

75.33 ± 9.93

70.5 ± 6.25

−

−1.3

5.1

74.33 ± 5.0

−

69.33 ± 5.09

−

66.33 ± 4.84

0.5 ± 1.22

***

−

ns

***

HCQ

HBA

HIN

65.83 ± 12.56

56.33 ± 17.82

46.67 ± 13.35

41.33 ± 14.87

49.17 ± 24.36

28.00 ± 11.30

11.4

24.2

37.2

44.4

33.9

62.3

61.0

56.0

88.8

83.8

84.3

16.6

36.9

72.6

53.8

48.0

19.83 ± 5.67

71.4

73.3

73.8

78.8

74.3

79.3

83.2

96.6

96.2

93.2

94.2

62.0

76.8

93.2

75.5

88.0

99.2

100

72.6

99

**

***

18.50 ± 11.39

0 ± 0

*

**

***

62.33 ± 10.95

16.1

41.9

4.4

18.16 ± 3.97

18.17 ± 3.97

0.67 ± 1.21

1.17 ± 2.04

*

**

***

**

***

HA

43.17 ± 11.05

14.67 ± 12.16

17.83 ± 15.48

14.33 ± 13.08

ns

***

HD

71.00 ± 5.20

98.2

100

98.0

100

99.0

98.5

97.0

99.0

*

***

HL

58.67 ± 6.34

52.67 ± 15.05

29.00 ± 15.72

21.0

39.1

61.0

62.8

66.0

70.9

−6.8

9.0

0 ± 0

*

**

***

BPA

29.00 ± 6.09

11.67 ± 0.58

2.33 ± 3.21

2.67 ± 2.08

4.67 ± 1.53

4.00 ± 3.60

1.33 ± 1.53

***

*

**

***

Indo

32.67 ± 14.74

0 ± 0

*

**

***

Aceclo

Diclo

Lico

27.67 ± 11.01

8.33 ± 2.08

12.00 ± 1.00

11.67 ± 1.15

0 ± 0

*

**

***

25.33 ± 2.52

0 ± 0

*

**

***

*

***

21.67 ± 10.07

0 ± 0

ns

***

HCQ + BPA

HCQ + Indo

79.33 ± 1.52

62.00 ± 16.82

26.33 ± 12.50

0.67 ± 1.15

1.00 ± 1.73

1.00 ± 1.00

2.00 ± 2.65

0.67 ± 0.58

*

***

69.00 ± 8.18

64.00 ± 1.00

29.67 ± 4.93

4.67 ± 3.51

17.00 ± 5.57

8.33 ± 6.43

*

**

***

HCQ + Aceclo 56.33 ± 7.58

24.3

37.2

29.2

20.33 ± 12.58

34.33 ± 10.69

38.67 ± 18.88

*

**

***

HCQ + Diclo

HCQ + Lico

46.67 ± 12.10

*

**

52.67 ± 8.73

#

Average of six readings ± SEM, statistically analyzed by two-way ANOVA followed by Bonferroni’s post-hoc test, P < 0.01as compared to HC,

P < 0.05, **P < 0.01, ***P < 0.001, ns: non-siginificant as compared to AAC

*

change in paw volume was recorded till 6 h instead of the

routine protocol of 3 h. Groups treated with HCQ showed

significant reduction of edema from 3 h after injection of car-

rageenan (53% inhibition of edema). At the end of 6 h, HCQ

exhibited 90% of inhibition of edema. All prodrug-treated

groups showed significant anti-edematous effect from 3 h with

at non-injected sites (fore-paws, ears, nose, and tail), a decrease

in weight, and immune response. In the last stages of arthritis,

rats become relatively immobile due to severe swelling in paw.

In the present investigation, effect of standard and test com-

pounds was studied on parameters like change in body weight,

percentage inhibition of paw edema and change in joint diam-

eter. Arthritic patients generally suffer from malnutrition and

this phenomenon is known as rheumatoid cachexia. In arthritic

patients, increased catabolism is observed. This increased ca-

tabolism and unmet needs of protein diet are the main reasons

behind weight loss and reduced lean body mass [28]. So, body

weight is one of the parameter evaluated while monitoring

severity of arthritis induced by CFA. Body weights of all

groups were measured before, in between and after induction

of disease with the interval of 4 days; the results of which are

shown in Table 4. Healthy control group showed constant in-

crease in body weight throughout the study. Decrease in body

weight of rats was observed in arthritic control group. The

treatment was started on the 12th day of the study by adminis-

tering test and standard compounds orally. HCQ-treated ani-

mals exhibited time-dependent decrease in body weight till

the 16th day of the study (from 204.0 ± 2.65 g to

188.33 ± 2.94 g). From the 16th day onwards, slight increase

in body weight was observed that increased to 197.17 ± 3 g on

the 28th day but did not reach the normal value. All prodrugs,

NSAIDs and physical mixture-treated groups significantly in-

creased the body weight except indomethacin and its physical

mixture with HCQ. HBA-treated groups showed remarkable

improvement in body weight.

5

5–84% of inhibition which resulted into complete inhibition

of edema (99–100%) at the end. HL showed maximum inhi-

bition of edema, i.e., 84% at 3 h. Licofelone (41%) and

aceclofenac (39%) showed significant analgesia at 1 h than

other NSAIDs. NSAIDs demonstrated 75–96% of inhibition

at 3 h with complete inhibition at the end. All physical mix-

tures showed 53–59% inhibition at 3 h and 99–100% inhibi-

tion at the end.

Anti-arthritic activity by Freund’s adjuvant-induced arthritis

Joint pain and chronic inflammation is evaluated by CFA-

induced chronic arthritis model, which is well correlated with

human body [25]. This model is characterized by features such

as consistent development and progression of disease and easy

monitoring of parameters like articular inflammation, bone de-

sorption, mild and disproportionate cartilage destruction [26].

The arthritogenicity of CFA largely depends on mycobacterial

DNA that binds to TLR9 and activates an adaptive immune

response [27]. A subcutaneous injection of CFA into the rat

paw induces inflammation as a primary lesion with a maximum

severity of 3–5 days. Secondary lesions occur after approxi-

mately 11–13 days, which are characterized by inflammation

Drug Deliv. and Transl. Res.

Table 4 Effect of prodrugs on body weight in CFA-induced arthritis

th

Groups

0

day

12 day

16 day

20 day

24 day

28 day

HC

198.83 ± 5.7

199.33 ± 7.0

204.0 ± 2.65

186.2 ± 3.49

199.8 ± 5.19

189 ± 5.10

208.67 ± 3.26

3.5 ± 1.54

214 ± 4.65

5.33 ± 1.39

222.33 ± 4.32

8.33 ± 0.33

226.5 ± 3.94

4.17 ± 0.38

231.83 ± 3.12

5.33 ± 0.82

Change in body weight

AC

#

177.67 ± 9.39

−12.33 ± 0.81

175.33 ± 9.71

−2.34 ± 0.32

188.33 ± 2.94

−1.84 ± 0.12

176.8 ± 3.13

0 ± 0.01

177.17 ± 8.95

1.84 ± 0.76

193.83 ± 6.14

16.66 ± 2.81

194.83 ± 3.43

1 ± 2.71

Change in body weight

HCQ

*

**

ns

*

190.17 ± 2.8

189.67 ± 3.2

1.34 ± 0.21

181.5 ± 3.89

4.7 ± 0.76

193.50 ± 3.2

197.17 ± 3.00

3.67 ± 0.21

205.3 ± 3.39

19 ± 0.69

Change in body weight

HBA

−5.16 ± 0.15

176.8 ± 3.12

3.83 ± 0.06

186.3 ± 4.08

4.8 ± 0.19

ns

**

ns

**

*

Change in body weight

HIN

−0.5 ± −0.08

*

***

*

186.7 ± 4.08

191.23 ± 6.26

4.53 ± 2.18

185 ± 3.90

192.2 ± 3.8

194.3 ± 1.97

195.2 ± 4.32

Change in body weight

HA

−3.8 ± 0.05

0.97 ± 2.43

188.8 ± 4.21

3.8 ± 0.31

2.1 ± 1.86

0.9 ± 2.35

*

**

*

***

183.5 ± 4.59

−0.5 ± −0.7

194.7 ± 5.95

201.2 ± 4.6

Change in body weight

HD

1.5 ± −0.69

5.9 ± 1.74

6.5 ± 1.33

202 ± 5.22

5 ± 3.59

*

**

***

*

****

196 ± 4.24

185.8 ± 3.65

−3.4 ± 0.11

176.3 ± 4.32

−1.9 ± 0.47

183 ± 7.32

191.5 ± 6.5

197 ± 8.81

5.5 ± 2.28

188.8 ± 5.56

4 ± 0.77

Change in body weight

HL

−2.8 ± 3.67

8.5 ± 0.79

184.8 ± 4.79

3.1 ± 0.71

ns

*

**

ns

**

183.2 ± 3.12

189.8 ± 3.45

186.7 ± 3.14

190.3 ± 3.78

197.5 ± 2.88

194.3 ± 1.97

197.2 ± 2.86

195.3 ± 3.01

203.8 ± 3.92

195 ± 3.16

181.7 ± 5.50

198.5 ± 5.16

9.7 ± 0.4

Change in body weight

BPA

5.4 ± 1.18

ns

*

168.4 ± 4.8

172.5 ± 6.0

178.6 ± 3.4

6.1 ± 2.6

187.6 ± 2.4

198.7 ± 4.5

Change in body weight

Indomethacin

−2.16 ± 1

173.7 ± 4.27

−5 ± 0.81

179.2 ± 5.98

−5 ± 0.49

4.1 ± 1.2

9 ± 1

11.1 ± 2.1

192.5 ± 2.59

6.8 ± 1.19

199 ± 4.20

6.7 ± 0.02

ns

**

ns

*

176.7 ± 6.50

180.7 ± 4.76

185.7 ± 3.78

5 ± 0.98

Change in body weight

Aceclofenac

3 ± 2.23

4 ± 1.74

ns

*

**

*

ns

**

182.8 ± 4.83

187.2 ± 4.45

4.4 ± 0.38

192.3 ± 4.18

5.1 ± 0.27

Change in body weight

Diclofenac

3.6 ± 1.15

*

***

*

***

185.5 ± 3.27

−3.7 ± 0.48

185 ± 3.35

−3.5 ± 0.69

185.7 ± 4.76

−4.6 ± 2.42

184.5 ± 3.39

−3.5 ± 1.84

184.5 ± 3.62

0.8 ± 1.12

189.3 ± 3.44

192.2 ± 3.31

2.9 ± 0.13

195.5 ± 2.59

201.2 ± 3.3

Change in body weight

Licofelone

3.8 ± 0.17

3.3 ± 0.72

5.7 ± 0.74

**

*

**

***

186.8 ± 6.18

1.8 ± 2.83

192.3 ± 4.68

5.5 ± 1.5

196.8 ± 2.48

4.5 ± 2.2

203.5 ± 3.5

6.7 ± 1.03

Change in body weight

HCQ + BPA

ns

183.2 ± 3.76

−2.5 ± 1.0

180.2 ± 4.31

−3 ± 0.55

185.3 ± 4.59

5.1 ± 0.28

192.7 ± 4.23

7.4 ± 0.36

Change in body weight

HCQ + Indo

*

ns

**

ns

**

*

ns

180.7 ± 2.80

−3.8 ± 0.59

185.2 ± 3.66

0.7 ± 0.04

176 ± 2.53

−4.7 ± 0.27

194 ± 3.52

8.8 ± 0.14

183.2 ± 3.66

7.2 ± 1.13

200 ± 4.98

6 ± 1.46

189.3 ± 3.44

6.1 ± 0.22

Change in body weight

HCQ + Aceclo

Change in body weight

HCQ + Diclo

*

***

205.7 ± 3.6

5.7 ± 1.37

ns

*

180.2 ± 2.93

−4.3 ± 0.28

186.8 ± 6.01

−5.9 ± 1.46

176.8 ± 2.64

−3.4 ± 0.29

183.8 ± 4.83

−3 ± 1.18

174.5 ± 3.78

−2.3 ± 1.14

180.5 ± 5.08

−3.3 ± 0.25

184.8 ± 4.59

10.3 ± 0.81

187.5 ± 5.89

7 ± 0.81

198 ± 4.50

13.2 ± 0.09

198.8 ± 1.47

11.3 ± 4.42

Change in body weight

HCQ + Lico

*

ns

*

199.3 ± 2.06

Change in body weight

#

Average of six readings ± SEM, statistically analyzed by two-way ANOVA followed by Bonferroni’s post-hoc test, P < 0.01as compared to healthy

control, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant as compared to AC

Change in paw volume is one of the important physiological

parameters for assessment of RA. Change in paw volume was

monitored by measuring paw volumes of rats on 0th, 12th, 16th,

in paw volume was observed till the 12th day. Though dosing

with test and standard compounds started on the 12th, significant

onset of inhibitory effect of HCQ on inflammation was demon-

strated only from the 20th day onwards and resulted into 75%

inhibition of edema on the 28th day. From the 20th to 28th day,

ameliorating effect of HCQ became more significant and result-

ed into moderate inhibition of paw swelling (75%). Overall ex-

tent of inhibition of inflammation produced by HCQ was the

lowest in all the treatment groups on the 28th day. Interestingly,

20th, 24th, and 28th day by using UGO Basile Plethysmometer.

Results of change in paw volume were given in Table 5. Set of

healthy animals showed no change in paw volume under the

study conditions. Arthritis control groups showed time-

dependent increase in paw volume of rats over the course of

study. In tests and standard-treated groups, significant increase

Drug Deliv. and Transl. Res.

% inhibition of edema

Table 5 Change in paw volume and % inhibition in CFA-induced arthritis

st

th

Groups

1 day

12 day

16 day

20 day

24 day

28 day

2

4th day

28th day

*

***

#

***

HC

0.01 ± 0.0

0.01 ± 0.00

0.01 ± 0.02

3.34 ± 0.09

3.01 ± 0.58

2.4 ± 0.23

0 ± 0

−

#

AC

0.30 ± 0.04 3.03 ± 0.02

4.92 ± 0.13

2.76 ± 0.39

2.1 ± 0.34

5.15 ± 0.28

5.98 ± 0.28

*

ns

**

*

***

HCQ

1.76 ± 0.08 2.79 ± 0.38

1.7 ± 0.02

1.5 ± 0.05

67

75

*

***

HBA

1.91 ± 0.76 2.78 ± 0.04

1.78 ± 0.06

1.99 ± 0.35

1.90 ± 0.23

1.89 ± 0.24

1.85 ± 0.30

1.63 ± 0.40

1.43 ± 0.38

0.74 ± 0.06

1.34 ± 0.19

0.54 ± 0.25

0.74 ± 0.81

0.29 ± 0.46

0.59 ± 0.33

0.41 ± 0.30

0.25 ± 0.39

0.39 ± 0.60

0.36 ± 0.08

0.27 ± 0.13

65.44

61.36

63.11

63.30

64.08

68.35

72.23

88.25

74

91

*

HIN

1.90 ± 1.00 2.57 ± 0.46

2.35 ± 0.42

2.50 ± 0.21

2.52 ± 0.32

2.47 ± 0.28

2.27 ± 0.45

2.60 ± 0.23

2.78 ± 0.12

2.44 ± 0.24

2.49 ± 0.27

2.35 ± 0.14

2.28 ± 0.33

1.98 ± 0.17

3.01 ± 0.26

2.96 ± 0.23

2.26 ± 0.31

2.33 ± 0.26

2.21 ± 0.33

2.11 ± 0.30

1.91 ± 0.48

1.83 ± 0.49

1.90 ± 0.13

2.33 ± 0.17

1.55 ± 0.23

1.78 ± 0.11

1.71 ± 0.34

2.13 ± 0.17

2.56 ± 0.23

2.84 ± 0.16

87.63

95.15

90.13

93.14

96

*

HA

2.37 ± .04

2.66 ± 0.24

ns

*

HD

1.97 ± 0.08 2.91 ± 0.29

1.93 ± 0.05 2.67 ± 0.22

1.76 ± 0.07 2.57 ± 0.30

1.02 ± 0.54 2.87 ± 0.66

0.91 ± 0.06 2.80 ± 0.40

0.96 ± 0.05 2.50 ± 0.22

0.75 ± 0.10 2.74 ± 0.12

0.86 ± 0.10 2.47 ± 0.08

0.71 ± 0.42 2.89 ± 0.51

HL

BPA

Indomethacin

Aceclofenac

Diclofenac

Licofelone

HCQ + BPA

HCQ + Indo

93.5

94

95.5

95

***

0.69 ± 0.20 ±

***

0.31 ± 0.27 ±

***

86.60

71

1.50 ± 0.08

0.30 ± 0.20

95

***

0.57 ± 0.63

0.32 ± 0.21

89

94.65

92.64

92

*

ns

*

***

HCQ + Aceclo 0.88 ± 0.09 1.83 ± 0.20

1.66 ± 0.22

0.44 ± 0.34

67.77

65.24

66.02

***

HCQ + Diclo

HCQ + Lico

0.93 ± 0.11 2.82 ± 0.16

1.01 ± 0.06 2.72 ± 0.13

1.79 ± 0.25

0.48 ± 0.38

**

***

1.75 ± 0.18

0.57 ± 0.32

90.5

#

Average of six readings ± SEM, statistically analyzed by two-way ANOVA followed by Bonferroni’s post-hoc test, P < 0.01as compared to healthy

control, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant as compared to AC

Table 6 Change in joint diameter in CFA-induced arthritis

st

th

Groups

1 day

12 day

16 day

20 day

24 day

28 day

% Change of joint diameter

2

4th day

28th day

*

**

***

#

***

#

***

#

***

#

HC

0.01 ± 0.01 0.01 ± 0.01

0.01 ± 0.01

3.16 ± 0.06

3.16 ± 0.13

3.23 ± 0.43

2.36 ± 0.23

3.23 ± 0.43

3.49 ± 0.10

3.52 ± 0.07

3.54 ± 0.19

2.86 ± 0.09

3.67 ± 0.10

3.71 ± 0.15

3.35 ± 0.31

3.72 ± 0.26

3.37 ± 0.16

3.44 ± 0.06

3.61 ± 0.13

3.66 ± 0.11

0.01 ± 0.01

3.17 ± 0.06

3.01 ± 0.12

2.48 ± 0.32

2.21 ± 0.14

2.48 ± 0.31

3.27 ± 0.15

2.83 ± 0.13

2.53 ± 0.18

2.42 ± 0.36

3.34 ± 0.11

2.29 ± 0.14

2.81 ± 0.10

3.40 ± 0.34

2.99 ± 0.15

3.39 ± 0.07

3.53 ± 0.12

3.60 ± 0.11

0.00 ± 0.00

3.19 ± 0.06

2.77 ± 0.11

2.21 ± 0.23

1.34 ± 0.20

2.20 ± 0.23

2.38 ± 0.20

1.72 ± 0.16

1.54 ± 0.29

1.65 ± 0.41

2.45 ± 0.31

1.90 ± 0.16

2.42 ± 0.17

2.35 ± 0.17

2.94 ± 0.15

2.78 ± 0.32

2.26 ± 0.11

2.38 ± 0.06

0.00 ± 0.00

3.19 ± 0.06

2.47 ± 0.10

1.14 ± 0.06

1.14 ± 0.12

1.14 ± 0.06

1.36 ± 0.36

0.75 ± 0.23

1.13 ± 0.15

1.20 ± 0.11

1.00 ± 0.16

1.40 ± 0.32

0.65 ± 0.26

1.40 ± 0.08

1.98 ± 0.33

1.65 ± 0.11

1.55 ± 0.36

1.67 ± 0.31

−

13.17

30.72

32

−

#

AC

3.06 ± 0.05 3.12 ± 0.06

ns

**

ns

**

ns

*

HCQ

3.06 ± 0.1

3.14 ± 0.12

22.57

64.26

57.37

76.5

ns

*

***

**

HBA

3.14 ± 0.06 3.40 ± 0.21

3.47 ± 0.23 3.16 ± 0.04

3.14 ± 0.05 3.40 ± 0.21

3.53 ± 0.17 3.62 ± 0.14

3.35 ± 0.12 3.57 ± 0.07

3.48 ± 0.15 3.59 ± 0.20

3.73 ± 0.20 3.85 ± 0.17

3.55 ± 0.25 3.89 ± 0.10

3.71 ± 0.19 3.66 ± 0.14

3.48 ± 0.13 3.67 ± 0.15

3.65 ± 0.27 3.75 ± 0.24

3.47 ± 0.27 3.60 ± 0.24

**

ns

*

***

*

HIN

HA

31.03

25.39

46.08

51.72

48.3

43.20

40.44

54.14

36.33

28

*

HD

**

***

**

***

**

HL

*

BPA

65.31

62.4

**

ns

**

*

Indomethacin

Aceclofenac

Diclofenac

Licofelone

HCQ + BPA

HCQ + Indo

68.65

56.11

79.62

48.24

32.26

42.60

46.33

57.65

ns

*

ns

***

HCQ + Aceclo 3.16 ± 0.05 3.39 ± 0.08

23

HCQ + Diclo

HCQ + Lico

3.43 ± 0.18 3.56 ± 0.14

3.49 ± 0.14 3.63 ± 0.10

22.2

36

#

Average of six readings ± SEM, statistically analyzed by two-way ANOVA followed by Bonferroni’s post-hoc test, P < 0.01 as compared to healthy

control, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant as compared to AC

Drug Deliv. and Transl. Res.

all prodrugs, NSAIDs and physical mixtures showed significant

onset of inhibitory effect on inflammation from the 16th day

onwards as against the 20th day by HCQ. All prodrugs produced

normal CRP level (30 ± 0.05) while arthritis control group ex-

hibited remarkable elevation in CRP levels (50.9 ± 0.1). HCQ

significantly arrested CRP levels (35.66 ± 0.56). Similarly,

prodrug-treated groups showed significant reduction in CRP

levels (32.1 ± 0.2–37.8 ± 0.05) as compared to NSAIDs

(38.9 ± 0.12–42.45 ± 0.09). Physical mixtures moderately re-

duced CRP (30.23 ± 0.6–35.67 ± 0.89). HL reduced CRP level

(32.1 ± 0.2) significantly out of all prodrugs.

88–95%, and NSAIDs and physical mixtures produced 90–96%

inhibition of paw volume on the 28th day of the study which

was significantly superior to HCQ (75%).

Change in joint diameter was monitored by measuring joint

diameter of rats on 0th, 12th, 16th, 20th, 24th, and 28th day by

using Vernier caliper. The results for change in diameter are

compiled in Table 6. Healthy control group showed no change

in joint diameter over a period of experiment. All groups

injected with CFA showed biphasic response where there

was a decrease in joint diameter from days 4 to 8; however,

this change was not significant. The joint diameter was max-

imum on the 12th day in all the CFA administered groups.

Decrease in joint diameter is the sign of decrease in the extent

of joint inflammation. HCQ showed significant inhibition of

joint diameter from the 24th day (13%) with 22.5% of inhibi-

tion on the 28th day. All prodrugs except HD showed signif-

icant inhibition from the 20th day study. On the 24th day, all

prodrugs continued to have 25 to 46% of inhibition of joint

diameter which culminated into 57–76.5% on the 28th day.

HL showed maximum inhibition (76.5%) on the 28th day. All

NSAIDs showed 56 to 79.6% of inhibition of joint diameter

on the 28th day. Physical mixture treated groups showed 32–

Hematological parameters The process of inflammation can

cause prominent changes in the blood count. The complete

blood count determines the WBC, RBC, hemoglobin (Hb),

hematocrit, several red blood cell indices and the platelet

count. Anemia, if often seen in arthritic patients mainly due

to gastrointestinal blood loss attributed to medications and

bone marrow changes due to inflammation [31]. Studies also

revealed that arthritic conditions causes mild to moderate

elevation of WBC count [32]. The platelet count is often

high in RA patients. Results of RBC, WBC, Hb, platelets

and ESR are graphically shown in Fig. 2e–i. RBC count was

significantly decreased in arthritic control (3.70 ± 0.30). All

prodrugs significantly improved RBC count (5.67 ± 0.13–6.

23 ± 0.7) as compared to HCQ (4.9 ± 0.2), NSAIDs (4.56 ± 0.

6–5.23 ± 0.67) and physical mixture groups. WBC count was

significantly increased in arthritic control (14.23 ± 0.56). All

prodrugs significantly decreased WBC count (9.00 ± 0.20–9.

5

7.6% of joint diameter inhibition on the 28th day.

43 ± 0.51) as compared to HCQ (11.9 ± 0.30), NSAIDs (10.

Biochemical parameters Effect of standard and test com-

pounds on biochemical parameters like ALT, AST, CRP, and

TPN in CFA-induced arthritic rats have been presented in Fig.

45 ± 0.51–11.1 ± 0.30) and physical mixture (10.45 ± 0.6–10.

73 ± 0.4) groups. Hb was significantly decreased in arthritic

control (9.21 ± 0.2). All prodrugs significantly improved Hb

(11.54 ± 0.23–12.45 ± 0.17) as compared to HCQ (10 ± 0.23),

NSAIDs (10.32 ± 0.82–10.89 ± 0.32) and physical mixture

groups (10.07 ± 0.33–11.76 ± 0.56). Platelets were

significantly increased in arthritic control (1789 ± 34). All

prodrugs significantly decreased platelets (1023 ± 45–

1090 ± 49) as compared to HCQ (1387 ± 45), NSAIDs

(1289 ± 40–1360 ± 47) and physical mixture groups

(1188 ± 34–1474 ± 49). Erythrocyte sedimentation rate

(ESR) is substitute marker for determination of extent of in-

flammation. ESR deviates due to increase in fibrinogen and

alpha proteins [33]. ESR was significantly increased in arthrit-

ic control (12.1 ± 0.3). All prodrugs significantly decreased

ESR (4.32 ± 0.3–7.01 ± 0.1) as compared to HCQ (5.45 ± 0.

37), NSAIDs (9.12 ± 0.21–9.9 ± 0.23) and physical mixture

groups (6.9 ± 0.08–8.9 ± 0.34).

2. In the present study, the possible adverse effect of prodrugs on

the liver was evaluated by performing assays for determination

of enzymatic levels of ALT and AST in CFA-induced arthritis

(Fig. 2a, b). In healthy control group, ALT and AST levels were

in normal range (44.0 ± 2.1 and 42.8 ± 1.6) which significantly

escalated in arthritic control group (148 ± 7.6 and 145 ± 7.6).

HCQ showed 130 ± 4.5 and 133 ± 4.5 ALT and AST values,

respectively. All prodrugs significantly reduced ALT and AST

levels (46 ± 5.6–56 ± 6.1 and 43 ± 5.6–58 ± 7.3, respectively).

HL remarkably reduced ALT and AST levels (46 ± 5.6 and

43 ± 5.6) out of all prodrugs. NSAIDs and physical mixtures

showed moderate effect on reduction of ALT and AST levels.

Inflammatory process involved in arthritis also affects body’s

protein level [29]. Hence, protein rich diet is recommended for

arthritic patients. Results of TPN are depicted in Fig. 2c. TP level

of the healthy control group was found to be (6.8 ± 0.03) which

was significantly decreased in arthritic control group (4.0 ± 0.8).

Prodrug-treated groups significantly increased TP levels

Liver accumulation One of the objectives of the present

study was to minimize accumulation of HCQ in non-

targeted sites like the liver. Therefore, we compared the extent

of accumulation of HCQ in the livers of sacrificed animals

treated with prodrugs, HCQ and physical mixtures, at the

end of CFA-induced arthritic study. The estimation was car-

ried out by HPLC and percentage of HCQ accumulated in the

(5.78 ± 0.65 to 6.4 ± 0.3) than HCQ (5.5 ± 0.15) and NSAIDs

(4.87 ± 0.12 to 5.8 ± 0.0.02). As a result of inflammatory stimuli,

glycoproteins such as C-reactive protein (CRP) are released in

blood which is a useful marker to assess severity of disease [30].

Results of CRP are summarized in Fig. 2d. Healthy control had

Drug Deliv. and Transl. Res.

a

b

2

1

00

50

00

200

150

100

50

#

ns

*

**

*

****

**

** **

**

****

*

**

*

**

***

*

**

*

**

***

**

*

**

***

*

***

5

0

Groups

ALT assay^a

AST assay^a

^c1₀

d ₁₅₀

*

**

8

6

4

2

0

***

*

**

*

**

*

** ***

***

100

*

** ***

***

#

***

#

**

****

**

***

5

0

*

** *** ***

**

***

*

**

***

Groups

Assay of C-reactive protein (CRP)^a

_A_s_s_a_y_o_f_t_o_t_a_l_p_r_o_t_e_i_n_n_u_t_r_i_e_n_ta

e

f

2

0

8

*

**

***

**

** ** **

*

6

4

2

0

***

** **

15

10

5

*

**

*

**

*

**

****

*

**

0

Groups

RBC count

WBC count

Fig. 2 Effect of prodrugs on various biochemical and hematological

parameters in CFA-induced arthritis. At the end of CFA model, blood

was withdrawn by retro-orbital puncture from rat and assayed for bio-

chemical (ALT, AST, TPN, CRP levels) and hematological (RBC, WBC,

platelets, Hb, ESR) parameters. After treatment with prodrugs (HBA,

HIN, HA, HD, HL), all changes in biochemical as well as hematological

parameters were restored back or compensated to normal level. a Average

of six readings ± SEM, statistically analyzed by one-way ANOVA

followed by Dunnett’s multiple comparison test, #P < 0.01 as compared

to HC, *P < 0.05, **P < 0.01, ***P < 0.001 as compared to AC

liver was calculated by taking into consideration the daily

dose of HCQ administered in CFA-induced arthritis model

Ulcerogenic potential Use of NSAIDs for the treatment of

RA involves long-term therapy and gastrointestinal intoler-

ance is the most common and serious adverse effect associated

with them. Gastric irritation, ulcers, perforation and hemor-

rhages are the prominent features of GI intolerance which

are ascribed to local and systemic effects of NSAIDs. The

local GI irritation is produced by free carboxylic group of

the NSAIDs. In the present work, –COOH group of selected

(Table 1). The results are depicted in Fig. 3. Prodrug-treated

groups showed half the concentration of released HCQ in the

liver (11–13.25%) as compared to HCQ (25.2%) and physical

mixture-treated groups (25–26%). So, significantly less accu-

mulation of HCQ in the liver (approximately 50% less) was

observed for prodrug-treated groups.

Drug Deliv. and Transl. Res.

g

h

2

1

000

500

000

1

5 ***

*

**

***

**

*

**

***

**

***

*

***

**

*

**

*

**

*

**

*

**

*

0

5

0

**

*

**

*** ***

*

**

*

** *** ***

500

0

Groups

Platelet count

Hemoglobin level

i

1

5

*

*******

**

0

5

0

**

*

**

*

***** ***

***

*

**

*

**

***

*

**

Erythrocyte sedimentation rate

Fig. 2 (continued)

NSAIDs was masked temporarily by conjugating it with –OH

of HCQ. Therefore, it was important to investigate gastro-

showed normal architecture of cells of rat ankle joint (Fig.

5A) (grade 0) with the presence of bundles of collagen

fiber and normal chondrocytes. Arthritic control (Fig. 5B)

(grade 3) showed prominent abnormalities and inflamma-

tory reactions like infiltration of immune cells, synovial

hyperplasia, pannus formation, cartilage erosion and in-

flammatory edema. HCQ-treated group (Fig. 5C) (grade

1) showed complete suppression of inflammatory infiltra-

tion, absence of signs of synovial hyperplasia and pannus

formation. All prodrug-treated groups (Fig. 5D-H) (grade

1) provided significant protection against the histopatho-

logical parameters. NSAIDs-treated groups (Fig. 5I-M)

(grade 0) showed absence of inflammatory infiltration,

signs of synovial hyperplasia and pannus formation and

comparable to healthy control and HCQ-treated groups.

Physical mixtures (Fig. 5N-R) did not provide protection

against any of the histological parameters against CFA-

induced arthritis model.

sparing ability of the synthesized prodrugs. At the end of the

th

2

8 day of CFA-induced arthritis model, rats were sacrificed;

the stomachs were scanned for ulcers and scored according to

Cioli et al. method (1979) [23]. The resulting ulcer indices are

shown in Fig. 4. All prodrugs reduced the ulcerogenic propen-

sity (4.9 ± 0.54 to 7.8 ± 0.09) of respective NSAIDs

(

8.2 ± 0.07 to 14.6 ± 0.06) and physical mixtures

10.4 ± 0.34 to 15.4 ± 0.08) extensively. Prodrug HL-treated

group showed the least ulcer index (3.5 ± 0.23) and HIN

showed the highest ulcer index (7.8 ± 0.09) out of all prodrugs

but comparatively less than respective NSAIDs and their

physical mixtures with HCQ. HCQ (5.34 ± 0.7) showed com-

paratively less ulcer index than all other groups.

Histopathology of rat ankle joints in CFA-induced arthri-

tis Histopathological system is used for evaluating an ef-

fective induction of disease and treatment against it [34].

th

On the 28 day of CFA model, rat ankle joints were re-

Radiological evaluation of rat paw Radiology is a useful

tool for evaluation of progression of arthritis. Joint inflamma-

tion is radiographically evaluated by parameters like joint

space narrowing, bone erosion and soft tissue swelling [36].

On the last day of CFA, model rats were sacrificed; X-rays of

ankle joints were recorded and analyzed with the help of

moved, decalcified and stained for histopathological study

with hematoxyline and eosin. Histopathological results

were graded according to Brenner et al. 2005 [35] grading

pattern. The observed histopathological changes of proxi-

mal tibio-tarsal joints of all groups are shown in Fig. 5. HC

Drug Deliv. and Transl. Res.

Fig. 5 Histopathology of ankle joints in CFA-induced arthritis model. A,

Healthy control. B, Arthritic control. C, Hydroxychloroquine. D, HBA.

E, HIN. F, HA. G, HD. H, HL I, Bi-phenyl acetic acid. J,

Indomethacin. K, Aceclofenac. L, Diclofenac. M, Licofelone. N,

Physical mixture of hydroxychloroquine and bi-phenyl acetic acid. O,

Physical mixture of hydroxychloroquine and indomethacin. P, Physical

mixture of hydroxychloroquine and aceclofenac. Q, Physical mixture of

hydroxychloroquine and diclofenac. R, Physical mixture of

hydroxychloroquine and licofelone. Arrow with circle at end indicates

lacunae containing chondrocytes, elbow double black arrow connector

indicates bundles of collagen fiber, black arrow indicates erosion of car-

tilage, line indicates pannus formation, elbow connector indicates inflam-

matory edema, solid black arrow indicates infiltration of inflammatory

cell, double black arrow indicates thinning of cartilage and U-turn black

arrow indicates hyperplasia of synovium

narrowing and extensive soft tissue swelling (grade 2). All

prodrug-treated groups showed mild erosion and joint space

narrowing (Fig. 6D-H). NSAIDs-treated groups exhibited

minimum narrowing of joint space and soft tissue swelling

with mild bone erosion (Fig. 6I-M) (grade 1). Treatment with

physical mixtures produced moderate bone erosion and min-

imum joint space narrowing without any effect on soft tissue

swelling (Fig. 6N-R) (grade 2).

Fig. 3 Accumulation of HCQ in the liver in CFA model. At the end of

CFA model, the livers were removed from sacrificed animals and assayed

for HCQ content. In prodrug-treated groups, HA and HL showed less

accumulation of HCQ was than plain HCQ and respective physical mix-

ture of HCQ and NSAIDs

Discussion

RA is a systemic inflammatory disorder characterized by sym-

metric, relapsing, or chronic musculoskeletal disability and

involves extra-articular complications. Currently, the primary

approach for clinical management of RA involves early inten-

sive medication with DMARDs that decreases disease activity

and improves physical function and quality of life.

Conventional DMARDs like MTX and HCQ are recommend-

ed as first line therapy for RA. NSAIDs, corticosteroids and

other non-pharmacological approaches are considered as ad-

junct to the therapy as it helps to control symptoms but does

not apparently modify the disease. Present work was focused

grading score reported by Pathak et al. 2010 [19]. The radio-

graphic features of the rat joints in CFA-induced arthritic mod-

el are shown in Fig. 6. Arthritic control (Fig. 6B) showed

ankylosis of soft tissue, severe bone erosion and narrowing

of joint space (grade 3) indicating bony destruction which was

absent healthy control group (Fig. 6A). HCQ-treated group

(

Fig. 6C) showed mild erosion, minimum joint space

Fig. 4 Ulcer index of groups in

CFA model. The stomach of

sacrificed animals at the end of

CFA model was scored for ulcers

by Cioli et al. method. Ulcer

index was seen to be higher in

NSAIDs and physical mixture

treated-groups than prodrugs;

hence, it was found to be safe. HL

prodrug showed least ulcer index

out of all prodrugs

Drug Deliv. and Transl. Res.

A

B

C

E

D

G

F

I

H

J

K

L

M

N

Q

O

P

R

Drug Deliv. and Transl. Res.

on latentiation of HCQ with NSAIDs as carriers by using

mutual prodrug concept.

Fig. 6 Radiology of rat ankle joints in CFA-induced arthritis. A, Healthy

control. B, Arthritic control. C, Hydroxychloroquine. D, HBA. E, HIN. F,

HA. G, HD. H, HL. I, Bi-phenyl acetic acid. J, Indomethacin. K,

Aceclofenac. L, Diclofenac. M, Licofelone. N, Physical mixture of

hydroxychloroquine and bi-phenyl acetic acid. O, Physical mixture of

hydroxychloroquine and indomethacin. P, Physical mixture of

hydroxychloroquine and aceclofenac. Q, Physical mixture of

hydroxychloroquine and diclofenac. R, Physical mixture of

hydroxychloroquine and licofelone. Arrow indicates severe bone erosion,

line indicates severe narrowing of joint space, elbow arrow connector

indicates ankylosis, arrow ending with circle indicates moderate bone

erosion, elbow arrow connector ending with circle indicates extensive

soft tissue swelling, dotted arrow indicates mild bone erosion, dotted line

indicates minimum narrowing of joint space and dotted elbow connector

indicates minimum soft tissue swelling

Chemistry

The formation of ester prodrugs was confirmed through

IR showing carbonyl stretching between 1745 and

−

1

720 cm . The absence of broad peak of hydroxyl group

−

1

of HCQ (3650 cm ) and shift in carbonyl peak of COOH

of NSAIDs towards higher frequency further confirmed

the formation of an ester. Chemical shifts of protons of

1

HCQ and NSAID backbones were confirmed by H–

NMR in which number of protons matched with the an-

1

3

ticipated structures. C–NMR confirmed formation of co-

valent ester linkage between one molecule of HCQ and

one molecule of NSAID. Mass spectroscopy also exhibit-

ed molecular ion peaks in accordance with the molecular

weights (530–696) of respective conjugates. Elemental

analysis results were within the permissible range that

confirmed molecular weights of conjugates.

the end of 7 h which could be ascribed to the steric hin-

drance around ester linkage.

In vivo behavior

Accurate estimation of in vivo pharmacokinetic properties and

factors affecting it are the guiding principles for successful

prodrug design. Appearance of prodrugs in blood at 1–1.5 h

after oral administration indicated fast absorption through the

stomach which was in accordance with their enhanced parti-

tion coefficients. There was consistent rise in the concentra-

tion of prodrugs in blood till 2.5–3.5 h that reached 3–5%

indicating some absorption of intact prodrugs from small in-

testine also. HCQ and NSAIDs appeared in blood from 3 h

onwards indicating hydrolytic activation of prodrugs in small

intestine. From these results, we hypothesized that prodrugs

must have been activated in small intestine by enzymatic

(esterase) and pH-dependent (pH 7.4) hydrolysis. The concen-

tration of intact prodrugs in blood started declining from 3 to

4 h and completely disappeared at 8–9.5 h. Concentration of

HCQ and NSAIDs reached a maximum of 40.5–49% at 10 h

and 53.4–66.8% at 8.5–10 h, respectively. Intact prodrugs and

NSAIDs were not observed in urine and feces samples where-

as 2.3–3.5 and 0.75–0.9% of HCQ was detected in 24-h

pooled samples of urine and feces, respectively.

Interestingly, many unknown metabolites were detected in

urine and feces which might be the result of metabolism of

HCQ and NSAIDs.

In vitro stability and kinetics

All prodrugs resisted acid-catalyzed hydrolysis when incubat-

ed with HCl buffer (pH 1.2) for 3 h as HPLC analysis did not

show any peaks of HCQ, NSAIDs, or unknown hydrolysis

product except the peaks of intact prodrugs. High chemical

stability of prodrugs in acidic environment would assist in

bypassing release of NSAIDs in the stomach that in turn might

prove to be beneficial in minimizing their direct contact-

irritant effect on the gastric mucosa and inhibition of the pros-

taglandins locally [37]. On the contrary, incubation with phos-

phate buffer (pH 7.4) over a period of 7 h hydrolyzed the

prodrugs (63–90% hydrolysis; Table 2) following first order

kinetics. Maximum hydrolysis was observed for HA (90%;

Table 2) as compared to other prodrugs. Half-lives of the

prodrugs were in the range of 137–315 min while the rate

−

3

constants were found to be between 2.2 × 10 and

3

−1

5

.0 × 10⁻min (Table 2).The ready release of HCQ and

NSAIDs in phosphate buffer indicated pH-dependent hydro-

lysis of prodrugs.

All prodrugs were stable in stomach homogenates that

justifies their gastro-sparing nature. Prodrugs were readily

hydrolyzed in intestinal homogenates by first order kinet-

ics. Due to the presence of enzymes, there was an increase

in the percentage of prodrugs hydrolyzed in intestinal ho-

mogenates (68–97%; Table 2) as compared to phosphate

buffer (pH 7.4). The rate constant values increased while

half-lives of the prodrugs decreased as shown in Table 2.

The extent of hydrolysis was maximum for HA (97%;

Table 2) which is in accordance with its hydrolysis in

phosphate buffer. The prodrug of HCQ and licofelone

Pharmacological screening

Analgesic activity

Acetic acid-induced writhing model involves chemically-

induced peripheral pain and analgesic activity of a test com-

pound is evaluated on the basis of its ability to decrease the

frequency of abdominal writhings in mice which are manifest-

ed as arching of back, extension of hind limbs and contraction

of abdominal muscles. Results of analgesia are shown in

(

HL) was an exception as only 68% was hydrolyzed at

Drug Deliv. and Transl. Res.

A

D

C

B

E

F

G

H

I

J

K

L

N

O

M

P

Q

R

Drug Deliv. and Transl. Res.

Table 3. HCQ is used in routine practice for treating synovitis

in inflammatory arthritides such as RA and has been shown to

be effective at reducing pain in pilot studies [38]. Analgesic

potential of NSAIDs is well documented in literature [39].

Therefore, it was important to assess the analgesic activity of

synthesized prodrugs and physical mixtures of parent com-

pounds. In acetic acid-induced writhing method, HCQ proved

its slow onset of action by showing inhibitory effect on latter

stages of writhes. All prodrugs showed higher analgesia than

HCQ till 120 min. It is notable that HA showed early onset of

analgesic activity with maximum analgesia by HA and HL as

compared to other prodrugs. The standard NSAIDs like

aceclofenac, licofelone, diclofenac, and indomethacin and

their physical mixtures with HCQ also produced significant

analgesic effect with an early onset of 60 min. From 120 min

onwards, there was a consistent rise in analgesic activity in all

groups treated with test and standard compounds. Initially

inhibition to NSAIDs at the end. As expected, NSAIDs-

treated groups showed rapid onset and complete inhibition

of edema at the end of the study and were able to inhibit the

second phase of inflammation which is in accordance with

literature reports [46]. It is a well-known fact that the second

phase of inflammation involves release of prostaglandins

which are the main causative agents of inflammation and are

liable for inhibition by most of steroidal and nonsteroidal anti-

inflammatory agents [47]. Groups treated with physical mix-

tures showed comparable inhibition of edema to NSAIDs-

treated groups.

Anti-arthritic activity

CFA-induced arthritis is the most extensively used chronic test

model in which the clinical and pathological changes are sim-

ilar with those seen in human RA. Chronic inflammation in

the CFA model is manifested as a progressive weight loss and

increase in the volume of the injected paw and joint diameter.

Inflammatory cytokines are released due to intra-articular in-

jection of heat-killed Mycobacterium tuberculae which affects

catabolism of arthritis patients. This observation is consistent

with the finding that inflammatory response impacts on food

and water intake of rat which leads to loss of weight [48].

After the start of treatment with HCQ, body weight of animals

(

after 60 min of administration), advantage of standard

NSAIDs over the prodrugs was evident but after 180 min,

equimolar doses of prodrugs produced significantly better an-

algesic activity over NSAIDs. At 240 min, all groups except

HIN exhibited complete inhibition of writhes.

Anti-inflammatory activity

th

Carrageenan, a phlogistic agent when injected into sub-plantar

region of rat paw, induces peripheral inflammation by activat-

ing innate immune pathway [40]. The resultant biphasic re-

sponse releases a cascade of inflammatory mediators that

cause pronounced inflammation leading to edema and red-

ness. The early phase releases histamine, serotonin and kinins

followed by various prostaglandins in the second phase which

serve as mediators of pain and inflammation [41]. Nitric oxide

has also been implicated in the inflammatory cascade respon-

sible for production of reactive oxygen species [42].

Carrageenan-induced paw edema model displays all biochem-

ical and cellular events involved in inflammation; hence, it

was chosen for studying onset, duration and mechanism of

action of prodrugs. Inflammation to joint is manifested as a

progressive increase in the volume of the injected paw which

was observed in arthritic control group (Table 1) indicating

proper induction of inflammation. Suppression of paw edema

at later stages signified slow onset of anti-inflammatory action

of HCQ which was in accordance with the literature reports

started increasing slightly from the 16 day but did not reach

the normal value (Table 4). These results indicated that HCQ

did not result in significant weight gain which is in accordance

with the findings of Newbould, Ward and Cloud, and Graeme

et al. (1966) stating that HCQ has insignificant effect on

weight gain in most of the patients of RA [49].

Indomethacin and its physical mixture showed no significant

increase in body weight which is in accordance with observa-

tions of Egan et al. who stated that toxic nature of indometh-

acin is responsible for weight loss in arthritic animals [50].

Significant increase in body weight of groups treated with

prodrugs, NSAIDs, physical mixtures except HIN, indometh-

acin, and its physical mixture conferred inhibition of inflam-

matory cytokines impacting food and water intake.

Injection of 0.1 ml CFA induces release of histamine, sero-

tonin, and prostaglandins. These inflammatory responses

cause activation of local pain receptors and nerve terminals

producing hypersensitivity at the area of injury. Increase in

paw edema at the infected site is the result of hypersensitivity

reaction. The increase in the extent of erythema and edema of

the tissue shows the severity of the inflammation [51]. Hence

monitoring the paw volume plays important role in assessing

the anti-arthritic activity [52]. As documented in literature,

HCQ started showing inhibition of paw volume on late phases

of chronic arthritis owing to its slow onset of action (Table 5).

Anti-inflammatory effect of HCQ is reported to involve its

impeding action on signaling of TLRs which in turn are im-

plicated in induction of inflammation by CFA [53]. Inhibition

[43]. According to some researchers, protection against in-

flammatory processes in joints associated with HCQ treatment

is due to oxygen radical scavenging effect [44, 45]. Anti-

edematous action of all prodrugs gradually and consistently

increased from 3 to 6 h resulting into complete inhibition of

edema at 6 h. Overall, the extent of inhibition of inflammation

offered by prodrugs was less at 3 h compared to NSAIDs or

their physical mixtures owing to slow release of HCQ and

NSAIDs from the prodrugs and culminated into comparable

Drug Deliv. and Transl. Res.

of edema produced by prodrugs was just moderately less than

other groups. NSAIDs-treated groups showed quick onset and

moderately superior of anti-edematous effect than all other

treatment groups at the end of the study as reported in litera-

ture [54]. All physical mixture-treated groups showed compa-

rable protection against inflammation to NSAIDs-treated

groups. Conjugation of HCQ with NSAIDs into mutual

prodrugs proved to be fruitful in terms of significantly better

anti-inflammatory/anti-arthritic activity than HCQ.

Joint diameter corresponds to the severity of inflammation

and arthritis. In severe arthritis, the diameter of tibio-tarsal

joint increases with the similar intensity as that of paw vol-

ume. So, this parameter plays important role in assessing the

anti-arthritic activity [52]. HCQ showed its onset of anti-

arthritic effect from the 24th day (13%) of treatment that re-

sulted in less reduction in joint diameter at the end of the 28th

day (22.5%) as compared to other groups (Table 6). Prodrug-

treated groups showed comparatively less gross reduction of

joint diameter as compared to NSAIDs but higher than HCQ-

treated group at equimolar doses. On the 28th day of the study,

prodrug HL, licofelone, physical mixture of HCQ and

licofelone (76.5, 79.6 and 57.6%, respectively) were found

to be most effective in reduction of joint diameter against all

treatment groups while prodrug HD, diclofenac and physical

mixture of HCQ and diclofenac (57.4, 56.1 and 46.3%, re-

spectively), produced moderate protective effect on joint di-

ameter. Effect of prodrugs on joint diameter was comparable

with respective NSAIDs and better than physical mixture but

they failed to abolish completely the swelling of the left ankle

joint.

One of the functions of the liver is to clear drugs from the

body, but excessive dose of NSAIDs like ibuprofen, aspirin,

naproxen, diclofenac, phenylbutazone, or DMARDs like

methotrexate and leflunamide can damage the liver, decrease

the rate of liver metabolism and the liver might also get in-

flamed and sensitive due to damage [55]. The important

markers of liver damage are liver enzymes like ALT/SGPT

and AST/SGOT whose levels are elevated in arthritis. In

healthy control group, ALT and AST levels were in normal

range. In arthritic group levels of ALTand ASTenzymes were

drastically increased due to inflammatory conditions such as

abnormal liver biopsies, mild portal chronic inflammatory in-

filtrate and fatty liver [56]. HCQ had negligible effect on

raised ALT and AST levels. These results are in accordance

with observations of Fries et al. stating that abnormalities of

transaminases enzyme are least frequent with treatment of

HCQ in arthritic patients [57]. Also it is reported that HCQ,

in spite of being a DMARD, has not been associated with

significant serum enzyme elevations during therapy of rheu-

matologic diseases [58]. Interestingly prodrug-treated groups

showed marked decrease in ALT and AST levels suggesting

recovery of damaged liver. So, the results of biochemical eval-

uation indicated that all prodrugs were hepato-protective and

minimized the severity of CFA-induced arthritis remarkably

as compared to standards. NSAIDs-treated groups showed

increased ALT and AST levels as literature reports shows that

NSAIDs induce liver damage leading to increased ALT/AST

levels [59]. In healthy control group, TPN was at normal level

(Fig. 2c). In the case of arthritic control, TPN level was de-

creased drastically. HCQ, NSAIDs, physical mixtures-treated

groups moderately improved TPN level whereas prodrugs

significantly improved TPN levels indicating significant pro-

tective effect on CFA-induced arthritis. The identification of

CRP is important because early detection of the disease will

allow for early treatment, which may limit functional disabil-

ity as a consequence of the disease. Arthritic control group

showed remarkable elevation in CRP levels while HCQ-

treated group showed decrease in CRP levels (Fig. 2d).

Blackburn et al. also observed drastic decrease in CRP levels

by HCQ [60].Similarly prodrug-treated groups showed signif-

icant reduction in CRP levels as compared to NSAIDs. These

results are in accordance with findings of Tarp et al. stating

that NSAIDs have no effect on the CRP in arthritic patients

[61]. Physical mixtures moderately reduced CRP.

Healthy control group exhibited normal levels of RBC,

WBC, platelet and Hb. RBC count and hemoglobin level were

decreased in arthritic control due to chronic inflammation

caused by induction of arthritis. WBC and platelet levels were

dramatically increased in arthritic control group. HCQ and

NSAIDs-treated groups showed slight increase in RBC and

Hb levels. Effect of HCQ on RBC/WBC count and Hb level

was in accordance with literature findings [62]. Prodrugs and

physical mixture-treated groups increased RBC count and Hb

level while decreased WBC and platelet counts effectively

than NSAIDs and HCQ-treated groups. Healthy control

showed normal ESR whereas arthritic control showed high

ESR (Fig. 2i) [33]. HCQ-treated group showed drastic de-

crease in ESR. This effect of HCQ on ESR levels is in accor-

dance with the reports in literature which state that HCQ re-

duces ESR levels in arthritic condition [8]. The elevated ESR

was significantly lowered by all the prodrugs; however, the

extent of lowering of ESR by NSAIDs and physical mixture-

treated groups was comparatively less than HCQ and

prodrugs.

Tanenbaum et al. (1980) have reported that after a long

term administration of HCQ, it binds to metabolically active

tissues like the liver, spleen, lungs and adrenal glands. This

effect is observed because of substantially high plasma protein

binding of HCQ. Tissue concentration of HCQ reaches 6000–

80,000 times the plasma concentration [63] and has narrow

therapeutic window [64]. Sequestration of HCQ in the liver

may lead to chronic hepatic diseases [65]. Interestingly,

prodrug-treated groups showed half the concentration of

HCQ in the liver as compared to HCQ and physical

mixture-treated groups. Significantly, less accumulation of

HCQ in the liver (approximately 50% less) observed for

Drug Deliv. and Transl. Res.

prodrug-treated groups might assist in lowering the extent of

side effects associated with long-term administration of HCQ

significant mitigation of other histopathological parameters.

NSAIDs-treated groups (Fig. 5) showed the absence of in-

flammatory infiltration, signs of synovial hyperplasia and

pannus formation (grade 0) which are commonly seen in the

developing RA. According to literature, NSAIDs inhibit ex-

pression of cytokines and prevent adhesion of monocytes ac-

tivating factor. This effect may inhibit migration of immune

cells to synovium and as a result, inhibition of infiltration of

inflammatory cells was observed [73]. Overall, NSAIDs pro-

vided significant protection against CFA-induced arthritis;

histologically, the results were comparable to healthy control

and HCQ-treated groups. Physical mixtures (Fig. 5) did not

provide protection against any of the histological parameters

against CFA-induced arthritis model (grade 3) and the results

were comparable with arthritic control.

Radiographic changes in RA conditions are useful diagnos-

tic measures which indicate the severity of the disease. Soft

tissue swelling is the earlier radiographic sign; whereas, prom-

inent radiographic changes like bony erosions and narrowing

of joint spaces can be observed only in arthritis. Healthy con-

trol (Fig. 6) group showed no signs of bone erosion, soft tissue

swelling and normal joint space (grade 0). Arthritic control

(Fig. 6) showed ankylosis of soft tissue, bone erosion and

narrowing of joint space (grade 3) indicating bony destruction.

HCQ-treated group (Fig. 6) showed mild erosion, minimum

joint space narrowing and extensive soft tissue swelling. All

prodrug-treated groups showed mild erosion and joint space

narrowing (Fig. 6). Except HA and HD, the remaining

prodrug-treated groups showed minimum soft tissue swelling.

NSAIDs-treated groups exhibited minimum narrowing of

joint space and soft tissue swelling with mild bone erosion

(Fig. 6). Treatment with physical mixtures produced moderate

bone erosion and joint space narrowing without any effect on

soft tissue swelling (Fig. 6).

[63, 64]. Simultaneously, the therapeutic window could be

broadened resulting in improved efficacy [64].

All prodrugs reduced the ulcerogenic propensity of respec-

tive NSAIDs and physical mixtures extensively due to tempo-

rary masking of free carboxylic group. Prodrug HL-treated

group showed least ulcer index. HCQ showed comparatively

less ulcer index than all other groups as treatment of HCQ is

not associated with complication of gastric ulceration [66, 67].

The results of in vitro and in vivo release kinetics also support

lowered ulcerogenic propensity of prodrugs. Stability of

prodrugs in the acidic environment of the stomach would re-

sist release of NSAIDs that are implicated in GI toxicity.

Healthy control showed normal architecture of cells of rat

ankle joint (Fig. 5) with the presence of bundles of collagen

fiber and normal chondrocytes and was graded 0 due to the

absence of inflammatory infiltrate, synovial hyperplasia,

pannus formation, cartilage erosion and edema. Arthritic con-

trol (Fig. 5) showed prominent abnormalities and inflamma-

tory reactions that were seen to progress quickly. Freund’s

adjuvant initiated production of arthritic synovial fibroblasts

that release chemokines responsible for infiltration of immune

cells in joint tissues. Arthritic group (grade 3) showed infiltra-

tion of inflammatory cells that indicated activation of

synoviocytes. Synovial hyperplasia occurs due to proliferation

and activation of synoviocytes [68] which was observed in

arthritic group. The presence of pannus formation was also

seen on the surface of cartilage of the joint. Pannus formation

is the result of collection of inflammatory cell and immune

complexes within synovial spaces and on cartilage of joints

[

(

69, 70]. This in turn produces matrix metalloproteinase

MMP). Cartilage erosion and inflammatory edema were seen

in histopathology of arthritic group which may be because of

MMP and cathepsin produced by synoviocytes [71].

Additionally, oxidative stress and activation of chondrocytes

plays a role in cartilage and bone erosion [72]. HCQ-treated

group showed complete suppression of inflammatory infiltra-

tion (grades 0–1). These results are seen in accordance with

findings stating that HCQ decreases cytokines like IL-6, IL-17

and IL-22 production by decrease in antigen presentation. So

these in turn, do not amplify infiltration of inflammatory cells

Conclusion

Two antiarthritic drugs acting by different mechanisms were

covalently conjugated successfully. Through these novel co-

drugs, problems of slow onset and accumulation of HCQ in

non-targeted sites and local gastric intolerance to NSAIDs

were well addressed. Marked improvement in quantifying pa-

rameters of analgesic, acute and chronic inflammation models

was observed in the prodrug-treated groups compared to HCQ

and physical mixtures. On overall comparison, HL and HA

were found to be more promising candidates among all other

prodrugs. Mutual prodrugs of HCQ with NSAIDs demon-

strate to provide wider spectrum of protection against inflam-

mation associated with RA than monotherapy comprising of

either HCQ or NSAID alone which is the most striking out-

come of this study. Mutual prodrugs of HCQ with NSAIDs

could be explored further as promising therapy for safe and

[

73]. HCQ-treated group exhibited comparable results to

healthy control characterized by absence of signs of synovial

hyperplasia and pannus formation which are generally seen in

the developing stage of RA (Fig. 5). All prodrug-treated

groups (Fig. 5) provided significant protection against the his-

topathological parameters. HL (Fig. 5) was observed to be the

most promising prodrug as its mitigating effect on inflamma-

tory infiltrate, synovial hyperplasia and pannus formation was

significantly superior to other groups and comparable to

healthy control group. HIN (Fig. 5) was the only prodrug that

provided amelioration of cartilage erosion along with

Drug Deliv. and Transl. Res.

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anti-inflammatory effect while simultaneously joint recon-

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6

00–5.

Acknowledgements One of the authors (PJ) is thankful to Basic

Scientific Research Scheme, University Grants Commission, New

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Article Doi

Novel drug delivery of dual acting prodrugs of hydroxychloroquine with aryl acetic acid NSAIDs: Design, kinetics and pharmacological study

DOI: 10.1007/s13346-017-0420-5

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